Axiom Genotyping Assay User Manual

User Manual
Axiom™ Genotyping Assay

For Axiom™ Genome-Wide and
Custom myDesign™ Array Plates
P/N 702830 Rev 4

September 2010
For research use only.
Not for use in diagnostic procedures.
Trademarks
Affymetrix®, GeneChip®, , NetAffx®, Command Console®, Powered by Affymetrix™, GeneChip-compatible™,
Genotyping Console™, DMET™, GeneTitan®, Axiom™ and myDesign™ are trademarks or registered trademarks of Affymetrix,
Inc. All other trademarks are the property of their respective owners.
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Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products, Affymetrix grants you a non-
exclusive, non-transferable, non-sublicensable license to use this Affymetrix product only in accordance with the manual and
written instructions provided by Affymetrix. You understand and agree that, except as expressly set forth in the Affymetrix
terms and conditions, no right or license to any patent or other intellectual property owned or licensable by Affymetrix is
conveyed or implied by this Affymetrix product. In particular, no right or license is conveyed or implied to use this Affymetrix
product in combination with a product not provided, licensed, or specifically recommended by Affymetrix for such use.
Patents
Arrays: Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,445,934; 5,744,305; 5,945,334;
6,140,044; 6,261,776; 6,291,183; 6,346,413; 6,399,365; 6,420,169; 6,551,817; 6,610,482; 6,733,977; 6,955,915 and D430,024 and
other U.S. or foreign patents. Products are manufactured and sold under license from OGT under 5,700,637 and 6,054,270.
Software products may be covered by one or more of the following patents: U.S. Patent Nos. 6,090,555; 6,611,767; 6,687,692;
6,829,376; 7,130,458; 7,451,047; and other U.S. or foreign patents.
Fluidics stations: Products may be covered by U.S. Patent Nos. 6,114,122; 6,287,850; 6,391,623; 6,422,249; and other U.S. or
foreign patents.
Scanners: Products may be covered by one or more of the following patents: U.S. Patent Nos. 6,141,096; 6,262,838; 6,294,327;
6,403,320; 6,407,858; 6,597,000; 7,406,391; and other U.S. or foreign patents.
Hybridization ovens: Products may be covered by one or more of the following patents: U.S. Patent Nos. 6,050,719; 6,386,749;
6,705,754; and other U.S. or foreign patents.
Copyright
© 2010 Affymetrix Inc. All rights reserved.
Contents
Chapter 1 About the Axiom™ Genotyping Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Overview of the Axiom Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Chapter 2 Genomic DNA Preparation and Requirements . . . . . . . . . . . . . . . . . . . . . . . 9

Sources of Human Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
General Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Genomic DNA Extraction/Purification Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Genomic DNA Cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Genomic DNA Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Chapter 3 Axiom™ Genotyping Assay:

Target Preparation with Biomek FXP Target Prep Instrument . . . . . . . . . . 15
Before Using the Biomek Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Stage 1 — DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Stage 2 — Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Stage 3 — Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Stage 4 — Preparation for the GeneTitan MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Chapter 4 Axiom Genotyping Assay:

Manual Target Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Before You Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Stage 1 — DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
Stage 2 — Fragmentation and Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .115
Stage 3 — Drying, Resuspension and QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .122
Stage 4 — Denaturation and Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .128
Stage 5 — Manually Preparing Ligation, Staining, and Stabilization Reagent Trays for the
GeneTitan MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .134
Chapter 5 Axiom™ Genotyping Assay:

Array Processing with the GeneTitan® MC Instrument . . . . . . . . . . . . . . . 149
Before Using the GeneTitan MC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
Stage 1 — Create and Upload Batch Registration File . . . . . . . . . . . . . . . . . . . . . . . . . . . .157
Stage 2 — Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .158
Status Window Prompts and Actions Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .170
Stage 3 — Ligate, Wash, Stain and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .172
Continuing the Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .181
Storing Hyb Trays for Rehybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .181
Shutting Down the GeneTitan MC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182
ii Axiom™ Genotyping Assay User Manual
Chapter 6 Automated Target Preparation for Processing Eight Axiom Array Plates Per
Week . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Thawing Frozen Plates of Amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185
Initial Target Prep Week — Biomek FXP Target Prep Express . . . . . . . . . . . . . . . . . . . . . . .186
Simultaneous Eight-Plate Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .192
Chapter 7 Automated Target Preparation for Processing Two Axiom Array Plates per
Week . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Overview of the 2-plate Workflow for Automated Target Preparation . . . . . . . . . . . . . . . .203
Target Prep and Array Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .204
Chapter 8 Manual Target Preparation for Processing Three Axiom Array Plates per
Week . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Overview of the 3-plate Workflow for Manual Target Preparation . . . . . . . . . . . . . . . . . . .209
Manual Target Prep and Array Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .212
Chapter 9 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

Biomek FXP Target Prep Express . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .217
GeneTitan Multichannel Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .217
Appendix A Fragmentation Quality Control Gel Protocol. . . . . . . . . . . . . . . . . . . . . . . 223

Protocol for Running a Fragmentation Quality Control Gel . . . . . . . . . . . . . . . . . . . . . . . .223
Appendix B Sample Quantitation after Resuspension. . . . . . . . . . . . . . . . . . . . . . . . . . 225

Protocol for Sample Quantitation after Resuspension . . . . . . . . . . . . . . . . . . . . . . . . . . . .225
Suggested Protocol for OD Quantitation Using the DTX 880 . . . . . . . . . . . . . . . . . . . . . . .227
If Performing Sample Quantitation on a Plate Reader Other than the DTX880 . . . . . . . . . .233
Appendix C Rehybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Protocol for Rehybridizing Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .235
Appendix D Registering Samples in Affymetrix GeneChip® Command Console . . . . . 237

Creating a GeneTitan Array Plate Registration File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .237
iii
Appendix E Deionization Procedure for GeneTitan Trays and Covers . . . . . . . . . . . . . 241
Appendix F GeneTitan® MC Instrument Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .254
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
iv Axiom™ Genotyping Assay User Manual
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1
About the Axiom™ Genotyping Assay

The first Genome-Wide Association study (GWAS) was published in 2005 (1) when individuals carrying
particular haplotypes of SNP rs380390 were found to have increased risk of developing age-related
macular degeneration, a study performed with the Affymetrix GeneChip Mapping 100K Array Set (2).
As of September, 2009, there have been over 400 peer-reviewed GWAS publications and over 1774 SNPs
have been implicated in human disease (3). Initial GWAS studies focused on the “common disease,
common variant” hypothesis (1) that held that haplotypes with a minor allele frequency (MAF) >5%
would show measurable contribution to human disease research.
Current research is shifting towards “complex disease, complex/rare variant” studies. As such, these
research projects require a broader catalog of human variation, such as is being generated by the 1000
Genomes Project (http://www.1000genomes.org). This project focuses on identifying alleles with a MAF
<5% across a broader spectrum of human ethnicities. In order to allow our customers to take advantage
of this novel and rare content for genome association and candidate gene studies in a cost effective and
timely manner, Affymetrix is introducing a new genotyping product line: the Axiom™ Genotyping
Solution.
The Axiom Genotyping Solution introduces a new genotyping technology platform that includes novel
assay biochemistry, array configuration and processing, and automated target preparation.
Affymetrix conducted an empirical screen of genomic content from dbSNP
(http://www.ncbi.nlm.nih.gove/projects/SNP/). The screen included markers from HapMap and the 1000
Genomes Project as well as other sources, using HapMap phase 3 samples and/or the original 270
HapMap samples. All of this information has gone into creating a proprietary Affymetrix database of
validated markers that can be interrogated using the Axiom Genotyping Assay.
Currently two arrays are available for use with the Assay:
Axiom Genome-Wide CEU 1 Array: optimized to provide high genetic coverage (r2 >= 0.8) in Northern
European populations (often referred to as Caucasian or CEU), with lower coverage in ASI and YRI
populations.
Axiom Genome-Wide ASI 1Array: optimized to provide high genetic coverage (r2 > 0.8) in East Asian
populations (specifically the HapMap populations of Han Chinese and Tokyo Japanese), with lower
coverage in CEU and YRI populations.
These arrays are the first in a suite of population specific peg arrays that leverages the content of this
proprietary Affymetrix database.
The Axiom Genotyping Assay interrogates biallelic SNPs and simple indels in a single, fully automated
assay workflow. Starting with genomic DNA, the samples are processed by performing either an
automatic or manual target prep protocol followed by automated processing of the array plates in the
GeneTitan MC Instrument.
Target prep uses methods including DNA amplification, fragmentation, purification and resuspension
of the target in hybridization cocktail.
P
Automated target prep uses the Beckman Coulter Biomek FX Target Prep Express liquid handler.
Manual target prep enables lab workers to perform the steps without the use of automation
equipment.
The hyb-ready targets are then transferred to the Affymetrix GeneTitan® Multi-Channel (MC)
Instrument for automated, hands-free processing (including hybridization, staining, washing and
imaging).
Cel files generated by the GeneTitan MC Instrument are processed using the Axiom™ Genotyping
Algorithm version 1 (Axiom GT1) available through Affymetrix Power Tools or Genotyping Console™
v4.0.
6 Axiom™ Genotyping Assay User Manual
In summary, the Axiom Genotyping Solution is a product line that provides catalog arrays that:
Are optimized for high genetic coverage of their population in question.
Utilize the latest content from dbSNP.
Provide highly automated, reproducible results suitable for GWAS.
Related Documentation
Affymetrix GeneTitan® Multi-Channel Instrument User’s Guide, P/N 08-0308
Affymetrix GeneTitan® Multi-Channel Instrument Site Preparation Guide, P/N 08-0305
Affymetrix GeneChip® Command Console™ 3.1 User Manual, P/N 702569
Genotyping Console User Guide
Axiom gDNA Sample Prep QRC P/N 792928
Axiom Automated Target Prep QRC Setup P/N 702831
GeneTitan MC Protocol for Axiom Array Plate Processing QRC P/N 702929
References
1. Manolio T.A. and Collins F.S.: The HapMap and Genome-Wide Association Studies in Diagnosis
and Therapy. Annu Rev Medicine 2009, 60:443–56
2. Klein RJ, Zeiss C, Chew EY, et al.: Complement factor H polymorphism in age-related macular
degeneration. Science 2005, 308:385–89
3. Hindorff LA, Junkins HA, Mehta JP, and Manolio TA.: A Catalog of Published Genome-Wide
Association Studies. Available at: www.genome.gov/gwastudies. Accessed 09/28/2009.
Overview of the Axiom Assay

Running the Axiom Assay requires the following sets of steps:
1. Genomic DNA Prep--Resulting in samples that meet requirements spelled out in Chapter 2, Genomic
DNA Preparation and Requirements on page 9.
2. Target Prep of the samples:
The target prep can be done using either:
Biomek Automation (see Chapter 3, Axiom™ Genotyping Assay: Target Preparation with Biomek
FXP Target Prep Instrument on page 15)

Manual Target Prep (see Chapter 4, Axiom Genotyping Assay: Manual Target Preparation on
page 95)
3. Array Processing, done with
GeneTitan MC Instrument
GeneTitan Instrument Control software
AGCC Portal software
See Chapter 5, Axiom™ Genotyping Assay: Array Processing with the GeneTitan® MC Instrument
on page 149.
A list of the required equipment and supplies for running the Axiom Assay using automated target
preparation and Manual target preparation can be found in the Axiom Site Prep Guide, P/N 702858.
Running Multiple Plate Workflows

Affymetrix provides workflows that allow you to run a set of samples and array plates through the
protocol using a minimum of personnel and a forty-hour week. The timing of steps is critical, whether
using automated target prep or manual target prep because of the following constraints:
Incubation after DNA Amplification is 23 hours
Chapter 1 | About the Axiom™ Genotyping Assay 7
Hybridization in GeneTitan is 23.5 hours.

Reagent trays for wash/stain/imaging must be prepared as Hybridization finishes
Limits to when a second hyb tray and array plate can be loaded into GeneTitan.
These limitations require careful timing.
The details are covered in:
Chapter 6, Automated Target Preparation for Processing Eight Axiom Array Plates Per Week on
page 183
Chapter 7, Automated Target Preparation for Processing Two Axiom Array Plates per Week on
page 203
Chapter 8, Manual Target Preparation for Processing Three Axiom Array Plates per Week on page 209
Safety Warnings and Precautions
CAUTION: All chemicals should be considered as potentially hazardous. We, therefore,

recommend that this product is handled only by those persons who have been trained in
laboratory techniques and that it is used in accordance with the principles of good laboratory
practice. Wear suitable protective clothing, such as lab coat, safety glasses and gloves. Care
should be taken to avoid contact with skin and eyes.
WARNING: The following components contain harmful or toxic ingredients:

Axiom Stabilize Soln: 8% Gluteraldehyde
Axiom HybSoln 2: 100% Formamide

Axiom Hyb Buffer: <55% Tetramethylammonium Chloride
As such we recommend the use of a fume hood when using these products during the Manual
Target Preparation protocol. In all cases customers should use adequate local and general
ventilation in order to minimize airborne concentrations.
Copies of the Material Safety Data Sheets for the kit components are available on the Affymetrix website
at www.affymetrix.com.

2
Genomic DNA Preparation and Requirements

The general requirements for genomic DNA (gDNA) sources and extraction methods are described in this
chapter. The success of this assay requires uniform amplification of the genome starting with relatively
intact gDNA. To achieve this, the gDNA must be of high quality, and must be free of contaminants that
may affect the enzymatic reactions to be performed.
For this protocol, you will use the Axiom Reagent Kit (96 reaction, P/N 901281). These kits contain a
tube labeled Axiom Reference Genomic DNA 103. This DNA meets the requirements outlined below,
and is included for use as a control. The size and purity of sample gDNA can be compared with those of
the control DNA to assess sample quality. The control DNA should also be used routinely as an
experimental positive control and for troubleshooting purposes.
Assay performance may vary for gDNA samples that do not meet the general requirements described
below. However, the reliability of any given result should be assessed in the context of overall
experimental design and goals.
The genomic DNA requirements and preparation are described in the following sections:
Sources of Human Genomic DNA
General Requirements on page 9
Genomic DNA Extraction/Purification Methods on page 11
Genomic DNA Cleanup on page 11
Genomic DNA Preparation on page 11
Sources of Human Genomic DNA

The following sources of human gDNA have been successfully tested in the laboratories at Affymetrix
for DNA that meets the above requirements.
Blood
Saliva
Cell line
Success with other types of samples will depend on quality (degree of degradation, level of purity, etc.)
and quantity of gDNA extracted.
NOTE: DNA derived from Formalin-Fixed Paraffin-Embedded (FFPE) blocks should not be used
with this assay.
General Requirements
Starting DNA must be double-stranded for the purpose of accurate concentration determination.
DNA must be of high purity.
DNA should be free of DNA polymerase inhibitors. Examples of inhibitors include high concentrations
of heme (from blood) and high concentrations of chelating agents (i.e., EDTA). The gDNA extraction/
purification method should render DNA that is generally salt-free because high concentrations of
particular salts can also inhibit enzyme reactions. DNA purity is indicated by OD260/OD280 and OD260/
OD230 ratios. The OD260/OD280 ratio should be between 1.8 and 2.0 and the OD260/OD230 ratio should be
greater than 1.5. We recommend that DNA samples that do not meet these criteria be cleaned up as
described under Genomic DNA Cleanup on page 11.
DNA must not be degraded.
The approximate average size of gDNA may be assessed on a 1% agarose gel using an appropriate size
standard control. Approximately 90% of the DNA must be greater than 10 Kb in size. Control DNA
can be run on the same gel for side-by-side comparison.
Assessing the Quality of Genomic DNA Using 1% Agarose E-gels

We recommend this quality control step to asses the quality of the gDNA prior to starting the assay.
Equipment and Reagents Recommended
Table 2.1 E-Gel® and reagents required

Item Supplier Part Number
Mother E-Base Device EB-M03
Daughter E-Base Device Life Technologies EB-D03

(formerly Invitrogen)
E-Gel® 48 1% agarose gels G8008-01
RediLoad™ 750026
E-Gel® 96 High Range DNA Marker 12352-019
Guidelines for Preparing the Genomic DNA Plate

Loading a DNA mass of 10 ng to 20 ng per well is recommended. If lower amounts are loaded,
omission of the loading dye is recommended in order to improve visualization. Loading ≥ 25 ng gDNA
per well can improve the image.
Add 3 µL of 0.1X of RediLoad dye to each sample.
Bring each sample to a total volume of 20 µL using H 2O (for example, if the volume of genomic DNA
is 5 µL, add 3 µL of RediLoad, and bring to 20 µL total by adding 12 µL of H2O).

Seal, vortex and spin.
To Run the E-Gel:

1. Power on for E-Base (red light).
2. Push the Power/Prg button to make sure the program is at EG mode (not EP).
3. Insert the 2*48 well 1% Agarose E-Gel into slot.
4. Remove 2 combs.
5. Load 20 µL from the above plate onto 2*48 well 1% agarose E-Gel.
6. Load 15 µL of diluted High Range DNA Marker (1:3 dilution or ~ 0.34 X from stock) into all marker
wells (as needed).
7. Fill all empty wells with water.
8. Adjust the run time to ~27 min.
9. Push the Power/Prg button again (it will change from red to green).
When run time is reached (the ladder band reaches the end of the lane), the system will automatically shut
off. The gel is then ready for imaging.
Chapter 2 | Genomic DNA Preparation and Requirements 11
Figure 2.1 Gel images showing intact gDNA and degraded gDNA.
Genomic DNA Extraction/Purification Methods

Genomic DNA extraction and purification methods that meet the general requirements outlined above
should yield successful results. Methods that include boiling or strong denaturants are not acceptable
because the DNA would be rendered single-stranded and can no longer be accurately quantitated using a
PicoGreen-based assay.
Genomic DNA Cleanup

If a gDNA preparation is suspected to contain inhibitors, the following cleanup procedure can be used:
1. Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at –20 °C), to gDNA.
2. Vortex and incubate at –20 °C for 1 hr.
3. Centrifuge at 12,000 x g in a microcentrifuge at room temperature for 20 min.
4. Remove supernatant and wash pellet with 80% ethanol.
5. Centrifuge at 12,000 x g at room temperature for 5 min.
6. Remove the 80% ethanol and repeat the 80% ethanol wash one more time.
7. Resuspend the pellet in reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).
(See the Axiom Site Preparation Guide P/N 702858 for reagents, equipment, labware and
consumables for Axiom Assay).
Genomic DNA Preparation

This step needs to be done before proceeding with the DNA amplification stages for either automated or
manual target prep.
The human genomic DNA (gDNA) you will process using the Axiom Assay should meet the general
requirements listed earlier in this chapter. A total of 200 ng gDNA is required.
To prepare gDNA:
1. Thaw Samples and Control
2. Quantitate and Dilute gDNA.
3. Aliquot the Diluted Samples and the Control
4. Freeze or Proceed
5. Create a Batch Registration File
Duration
Thirty minutes to an hour for reagents to thaw and half an hour for setup.
Equipment, Consumables and Reagents Required
Equipment and Consumables

The equipment and consumables listed in Table 2.2 are required for this stage.
Table 2.2 Equipment and consumables required for Genomic DNA Preparation.
Quantity Item
As required Adhesive seals for plates
1 Ice bucket, filled with ice
1 each Pipettes:
Single-channel P10 or P20
Optional: multi-channel P10 or P20
As required Pipette tips
1 Plate, deep well:

For Automated Target Prep: Beckman Deep Well Titer, polypropylene; P/N 267007
For Manual Target Prep: ABGene 96 Square Well Storage; AB-0932
1 Plate centrifuge
1 Plate spectrophotometer
(required only if no OD measurements available for samples)
1 Vortexer
Reagents
The reagents listed in Table 2.3 are required for this stage.
Table 2.3 Reagents required for Genomic DNA Preparation.

Reagent Supplier Part Number
From the Axiom Reagent Kit
Axiom Reference Genomic DNA 103 (use as a positive control) — —

located in Module 1, –20 °C
User-supplied
Reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) USB 75793
TEKnova T0223
1. Thaw Samples and Control

Thaw the components listed below to room temperature:
gDNA samples
Axiom Reference Genomic DNA 103 (from the Axiom Reagent Kit)
To thaw, either:
Place items on benchtop for one hour
Thaw in a water bath:
A. Fill a small plastic dish with Millipore water. Do not overfill as the level of the water should not
overflow when the sample tubes or plates are placed in the bath.
B. Thaw the sealed sample plate and Reference sample for a half-hour.
Chapter 2 | Genomic DNA Preparation and Requirements 13
C. Wipe off the sample plate after removing and before removing the lid to minimize the chances that
the water will enter the well and cause contamination or reaction failure.
2. Quantitate and Dilute gDNA

To quantitate and dilute the gDNA:
1. Gently vortex (50% maximum) and spin the gDNA and Reference Genomic DNA 103.
2. Recommendation: quantitate each sample (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit).
3. Dilute each sample to a concentration of 10 ng gDNA/µL using reduced EDTA TE buffer.
NOTE: Do NOT dilute the Reference Genomic DNA 103 control from the Axiom Reagent
Kit. It is already at a working concentration.
4. Seal, vortex and spin.
3. Aliquot the Diluted Samples and the Control

Next, the samples and control are place in one of the following deep well plates for target preparation:
For Automated Target Prep: Beckman Deep Well Titer, polypropylene; P/N 267007
For Manual Target Prep: ABgene 96 Square Well Storage; AB-0932
Aliquot diluted samples and Reference Genomic DNA 103 to the selected deep well plate as follows:
1. 20 µL of each diluted gDNA sample (this should be the equivalent of 200 ng of gDNA).
2. 20 µL of the Reference Genomic DNA 103 control.
We recommend including at least one positive control on each plate.
3. Seal and spin.
At this point you can:
Store the sample plate at –20 °C, or
Proceed to DNA Amplification for:
Automated Target Prep (see Chapter 3, Axiom™ Genotyping Assay: Target Preparation with Biomek
FXP Target Prep Instrument on page 15)

Manual Target Prep (see Chapter 4, Axiom Genotyping Assay: Manual Target Preparation on
page 95)
NOTE: You can leave the gDNA sample plate at room temperature if proceeding immediately
to DNA Amplification.
5. Create a Batch Registration File
IMPORTANT: It is very important to create and upload a GeneTitan Array Plate Registration
file with your sample information prior to loading the Array Plate and hyb tray in GeneTitan.
We recommend that you create (but not upload) this file at the same time you prepare your
plate of genomic DNA. When your samples are ready for hybridization, you will scan the array
plate barcode and upload the file to Affymetrix GeneChip Command Console (AGCC).
GeneTitan Array Plate Registration files contain information that is critical for:
Data file generation during imaging.
Tracking the experimental results for each sample loaded onto an array plate.
Detailed instructions for creating this file are located in Appendix D, Registering Samples in Affymetrix
GeneChip® Command Console on page 237. See also Figure 2.2 for a screen shot showing an example
of a batch registration file.
1. Open AGCC Portal > Samples, and select:
A. GeneTitan Array Plate Registration.
B. The array plate format.
C. Click Download.
2. Enter a unique name for each sample and any additional information.
3. Save the file.
The array plate barcode will not be scanned until you are ready to load the array plate and samples onto
the GeneTitan MC Instrument for processing.
Figure 2.2 Example of a batch registration file

3
Axiom™ Genotyping Assay:
Target Preparation with Biomek FXP Target Prep Instrument
The Axiom™ Genotyping Assay with automated target preparation is designed for processing 96 samples
at a time on Axiom™ Genome-Wide and Custom myDesign™ Array Plates. The protocol is performed
on two instruments:
Part 1: Target preparation is performed on the Biomek FXP Target Prep Express
Part 2: Array processing is performed on the GeneTitan® Multi-Channel (MC) Instrument
A list of all equipment and resources for the Axiom Genotyping Assay with Automated Target Prep is in
the Axiom Site Preparation Guide P/N 702858.
This chapter includes instructions for Part 1: target preparation. These instructions are presented as
follows:
Before Using the Biomek Workstation on page 15
Seal, Vortex and Spin on page 15
Breaking the Light Curtain on page 16
Plate Centrifuge on page 17
Labeling GeneTitan Hybridization and Reagent Trays on page 35
Pipette Tip Usage on page 17
Set the Biomek Software Default Settings on page 17
Equipment, Consumables, Labware and Reagents Required on page 22
Reagent Block Template on page 40
Related Biomek FXP Target Prep Express Documentation on page 40
IMPORTANT: Before proceeding to DNA Amplification, do the gDNA preparation described

in Chapter 2, Genomic DNA Preparation and Requirements on page 9.
Stage 1 — DNA Amplification on page 40

Stage 2 — Fragmentation and Purification on page 53
Stage 3 — Resuspension and Hybridization Preparation on page 62
Stage 4 — Preparation for the GeneTitan MC Instrument on page 72
Before Using the Biomek Workstation
Seal, Vortex and Spin

Unless otherwise stated in the protocol, follow the guidelines below when instructed to seal, vortex and
spin plates or reagent tubes for the Biomek FXP Target Prep Express portion of this assay.
Seal plates — we recommend using MicroAmp Clear Adhesive Films to seal your plates.
IMPORTANT: Always ensure that your plates are tightly sealed. A tight seal will prevent
sample loss and cross-well contamination.
Vortex:
Reagents 3 times, 1 sec each time at the maximum setting.
Plates 1 sec each corner, and 1 sec in the center at the maximum setting.
Spin — when instructed to spin plates or reagent vials, follow these guidelines unless otherwise
instructed (for example, when centrifuging and drying pellets, 5. Centrifuge and Dry Pellets on
page 61).
Plates:
• Spin at 1000 rpm for 1 min at 4 °C.

• Do not spin for more than 1 min.
Reagent Vials: 3 sec
Breaking the Light Curtain

For your safety, the Biomek FXP Target Prep Express is designed to immediately halt all movement when
the light curtain is broken.
Light Curtain Broken While Running the Assay

For your safety, the Biomek FXP Target Prep Express is equipped with a light curtain (Figure 3.1). The
light curtain senses when an object (such as a hand or an arm) enters the space surrounding the deck.
When this curtain is broken, all movement on the deck halts until the user either clicks OK to resume the
activity that was taking place, or aborts the activity. Incubation timers are not interrupted.
Figure 3.1 Prompt displayed when the light curtain is broken during the process referred to as Homing All Axes.
The light curtain covers the front of the

instrument. The sides are protected by
Plexiglas. Both areas are shown as red
in this figure. Any penetration of the
light curtain from outside the deck
halts all movement of the workstation.
To resume activity on the deck, click
OK. To abort the step or other activity,
click Stop on the toolbar.
Chapter 3 | Axiom™ Genotyping Assay: Target Preparation with Biomek FXP Target Prep Instrument 17
Pipette Tip Usage
Table 3.1 Pipette tip usage for one full run of the Axiom™ Genotyping Assay on the Biomek Workstation
Multi-Channel Multi-Channel Span-8 Span-8
Step P50, Pink AP96 P250, Aqua Span P250, Span P1000,
96 tips, P/N 96 tips, P/N Green Yellow
A21586 717253 96 tips, P/N 96 tips, P/N
379503 987925
96 rxns 96 rxns 96 rxns 96 rxns
DNA Amplification — 96 tips — 34 tips
Fragmentation — 96 tips 24 tips 20 tips
Resuspension and Hybridization Preparation 96 tips 96 tips 17 tips 15 tips
Preparation for GeneTitan™ — 96 tips 26 tips 96 tips

Denature samples
Transfer denatured samples to Hyb Tray
Prepare GeneTitan reagent plates
TOTAL 96 tips 384 tips 67 tips 165 tips
Plate Centrifuge
One plate centrifuge is required for the Axiom™ Genotyping Assay. Refer to the Axiom Site Preparation
Guide, P/N 702858, for an appropriate plate centrifuge that can be used with the Axiom Genotyping
Solution When centrifuging and drying pellets as instructed under 5. Centrifuge and Dry Pellets on
page 61, the centrifuge must be able to spin down plates at:
rpm: 4000
rcf: 3200
temperature: 4 °C
In addition, the bottom of the rotor buckets should be soft rubber to ensure that the deep well plates do
not crack. Do not spin plates in metal or hard plastic buckets.
NOTE: Refer to the Axiom Site Preparation Guide, P/N 702858, for an appropriate plate
centrifuge that can be used with the Axiom Genotyping Solution.
Set the Biomek Software Default Settings

Typically you will select the default settings for the Biomek software once. The settings you select will:
Determine the default step selected when the user prompt is displayed at the start of each run.
Determine which process controls (if any) that will be run during stages 2 and 3 (Fragmentation and
Resuspension). The process controls include:
Preparation of sample dilution plates for OD and gel analysis during Resuspension and Hyb Prep.
The dilution plates are taken off-deck. One is used for OD quantitation to evaluate DNA mass; the
other is used to check fragment size.
Prompt you to manually take samples for DNA quantitation prior to fragmentation.
To select Biomek Software default settings:

1. Launch the Biomek Software.
2. Open Project > Open Project > Axiom Target Prep and click OK (Figure 3.2).
Figure 3.2 Opening the Axiom Target Prep project
3. Open File > Open to display the Open Method window (Figure 3.3).
Or click the Open Method icon
4. Select Axiom Target Prep and click OK.
.
Figure 3.3 Opening the Axiom Target Preparation method
The Axiom Target Prep project folder

must be displayed in this menu.
5. In the left pane of the window, select Axiom Target Prep (Figure 3.4).
The default settings window is displayed on the right.
Figure 3.4 Click Startup Dialog to open the default settings window
The selections made in

this box, Choose default
settings:, are displayed
each time this window
appears.
The options in the

Select preferences box
must be selected prior
to starting a run.
You are not prompted
to select any
preferences at start up.
6. Select your default settings.

Choose defaults settings — these settings can be changed at the start of each step
Which array plate format?
NOTE: At present, the Axiom Genome-Wide Assay supports only the 96-array plate format.
Select 96.
Which step do you want to run?
The step that you choose will be the default setting for the runtime prompt. The actual step selected
when the runtime prompt is shown reflects the state of the last completed run of the Axiom Target
Preparation method. For example, if DNA Amplification was the step completed in the last run,
then the step chosen in the subsequent run would be Fragmentation (the next step in the process).
If there is no record of the previous run, then the default choice specified is selected.
Select preferences — These settings are displayed in this window only. You must select/deselect here.
QC check points
Prompt for manual DNA quantitation before fragmentation — the workstation will pause
following inactivation of the DNA amplification reaction to allow you to manually remove an
aliquot of each sample for off-line (manual) DNA quantitation. This extra quality control step
is available for troubleshooting the DNA amplification reaction.
Prepare plates for gel QC and OD after resuspension — the workstation will prepare two plates
of resuspended samples properly diluted for the fragmentation gel QC and OD quantitation
process control checks. See Appendix A, Fragmentation Quality Control Gel Protocol on
page 223 and Appendix B, Sample Quantitation after Resuspension on page 225 for
instructions and result assessment guidelines.
IMPORTANT: For troubleshooting and support purposes, we strongly recommend that

you perform the gel QC and OD quantitation process controls after resuspension.
Custom run options

Run method in test mode — select this option to skip all of the incubation timers in a step. If
selected, a prompt is displayed asking you to confirm that you want to run a step in test mode
(Figure 3.5). Use this option to perform runs using water only, not actual reagents or samples.
CAUTION: Never process samples in test mode. The assay will fail; all of your
samples and reagents will be lost.
Figure 3.5 Prompt displayed when the custom run option, Run method in test mode, is selected.
Deck configuration options

TRobot- select this option to perform the denaturation of the Axiom Hyb Ready Plate on the
integrated Biometra TRobot 96 thermal cycler

PTC - select this option to perform the denaturation of the Axiom Hyb Ready Plate on the
integrated Bio-Rad PTC-200 thermal cycler

No integrated thermal cycler - select this option to perform the denaturation of the Axiom Hyb
Ready Plate on an off-deck thermal cycler or if your Biomek does not have an integrated thermal
cycler. A list of thermal cyclers that have been verified with the assay can be found below. When
selecting this option, select the appropriate plate type that should be used for the Hyb Ready
Plate.
Select the Bio-Rad Skirted option when using the HSP 9631 plate for the PTC-200 or the Bio-
Rad Tetrad 0240G thermal cycler.

Select the Bio-Rad Semi-Skirted on Costar Round Bottom option for the ABI 9700 or the ABI
2720 thermal cycler. The Bio-Rad HSS 9601 plate must be stacked onto the Costar Round
Bottom plate from Corning (VWR International P/N 29442-392, E&K Scientific: EK
680568, Corning Mfg PN 3795) for the robot to prepare the Hyb Ready Plate.
We have verified the performance of this assay using the Bio-Rad PTC-200/PTC-200G and Biometra
TRobot 96 on the Beckman Biomek Target Prep Express liquid handler. We have also verified the
performance of this assay using the following off-deck thermal cyclers:
Bio-Rad PTC-200G
Biometra TRobot 96
ABI 9700 with a gold, silver or aluminum block

ABI 2720
Bio-Rad MJ 0240G
The performance of this assay has not been verified with other thermal cyclers.
Use of other thermal cyclers may result in assay failure and may violate the Axiom Array and Reagent
replacement policy.
IMPORTANT: The default settings you select here will be displayed each time the Axiom
Target Prep window is displayed.
The options displayed in the Startup Dialog box as shown in Figure 3.4 on page 19 must
be selected prior to starting a run. These settings are not prompted for at runtime.
The thermocycler needs to be programmed with the “Axiom Denature” protocol:

A. 95 °C 20 min
B. 48 °C 3 min
C. 48 °C hold
Use the heated lid option when setting up or running the protocol.
WARNING: Evaporation during denaturation can negatively impact assay performance. Use
the recommended thermal cycler consumables and sealing film to eliminate condensation
and evaporation. The arched, auto-sealing metal plate with P pads as shown in Table 3.2 on
page 22 should be replaced after use as per the manufacturers recommendation.
7. Click Start at the top of the left pane to close the default settings window.
Equipment, Consumables, Labware and Reagents Required
Labware used on the Biomek Workstation deck
Table 3.2 Labware used on the Biomek Workstation deck

Labware Supplier and Part Labware Image
Number
Biomek AP96 – P250 Pipette Tips Beckman Coulter

(aqua box; pre-sterile, barrier) P/N 717253
Biomek Span P1000 Pipette Tips Beckman Coulter

(yellow box; pre-sterile, barrier, P/N 987925
conductive)

P/N 379503
(green box; pre-sterile, barrier)

Number

P/N A21586
(pink box; pre-sterile, barrier)
BIO-RAD Hard Shell 96-well plate BIO-RAD

(available in multiple colors) P/N HSP9631
Lid, metal BIO-RAD Front view

(arched, auto-sealing with P pads) P/N MSL-2032 and
P Pad P/N: MSP-1003
Side view
Beckman Deep Well Titer Plate Beckman Coulter

(polypropylene) P/N 267007

Number
ABGene 96 Square Well Storage Thermo Scientific

Plate P/N AB-0932
(2.2 mL)
OD Plate, UV E & K Scientific

EK-25801
Frame for Reservoirs Beckman Coulter

Frame
P/N 372795

Number
Plate, Costar Brand Serocluster round VWR International P/N

bottom plate from Corning 29442-392
Note: this consumable is required if E&K Scientific EK
using an off-deck ABI 9700 or ABI 680568
2720 thermal cycle Corning Mfg PN 3795
Hard-Shell Full-Height 96-Well Semi- Bio-Rad HSS-9601

Skirted PCR Plate required only if using
off-deck ABI 2720 or ABI 9700 Thermal
cyclers
The ABI 9700 and the ABI 2720 use the semi-skirted 96-well plates
(PN HSS-9601) stacked on a Costar brand Serocluster 96-well
Round Bottom Microtitration plate as shown in the figure below
A 96-well semi skirted PCR plate stacked on a Costar Branded
Round Bottom 96 well plate.
Half Reservoir Beckman Coulter

Half module, 75 mL capacity P/N 372786

Number
Quarter Reservoirs Beckman Coulter

Undivided Divided by width
Quarter module, 40 mL capacity
Quarter module divided by width,

40 mL capacity 19 mL capacity
P/N 372790 (40 mL)
19 mL capacity each receptacle
P/N 372792 (19 mL)
Reagent block, chilled to 4 °C Beckman Coulter

A1 Metal posts on block circled in red.
P/N A83054
Reagent block template Contact Affymetrix Template on reagent block. Metal posts on block
circled in red.
(designed specifically for use with the
Axiom Reagent Kit)

Number
24-Position Tube Rack Beckman Coulter

with one 11 mm tube insert in position P/N 373661 (rack) Tube insert
A6. P/N 373696 (insert) A6
Adaptor, Deep Well Plate Beckman Coulter The metal block is the adaptor.
(installed on the Shaking Peltier) P/N A83050
This adaptor is typically installed by a

Beckman Coulter field service technician
during new system installation or a
system upgrade. Ensure that you have
one of these adaptors on the deck prior
to running this assay.
GeneTitan MC Consumables
All consumables for the GeneTitan MC Instrument are provided by Affymetrix. The following table
provides guidance on the consumables that are shipped with the Array Plate.
IMPORTANT: All covers must have barcodes. Discard any cover without a barcode.
Table 3.3 Axiom GeneTitan Tray consumables

Item Part Number Labware Image Information
HT Array All array plates The HT Array Plate

Plate have the PN shipping package
Package 202091 etched includes the following:
on the on the The function of the clear
plastic plastic cover for the
Array Plate is to protect
the Array Plate during
transport. You can
discard this after
removing the Array
Plate.
The Array Plate must be
protected at all times
from damage or
exposure to dust. The
Array Plate must be in
the Blue Array Plate
Protective Base at all
times.
The Blue Array Plate
Protective Base in the
package must be used
to protect the Array
Plate from damage.
Table 3.3 Axiom GeneTitan Tray consumables (Continued)

The HT Scan Tray

shipping package
includes the following:
The HT Scan tray
includes a Scan Tray
cover. The tray cover
should be used to cover
the scan tray before
placing the tray in the
GeneTitan MC
Instrument. The Tray
cover should be de-
ionized before use. See
the section Appendix E,
Deionization Procedure
for GeneTitan Trays and
Covers on page 241 for
the anti-static
procedure.
The Scan tray must be
from damage or
Scan Tray must be in the
Black Plate Cover at all
times.
The Black Scan tray
cover in the package is
used to protect the scan
tray glass from damage.
The black scan tray is
distinct from the Blue
Array Plate Protective
Base and must not be
used with the Array
Plate. Remove the
protective base from the
scan tray before loading
the scan tray with the
scan tray cover
Black Scan PN 202096 The Black Scan tray

Tray cover in the package is
protective used to protect the scan
base tray glass from damage.
used with the Array
Plate.
Remove and discard the

Scan Tray Scan Tray 501006 The GeneTitan Scan tray

with cover Cover 202757 must be loaded with the
scan tray cover into the
GeneTitan instrument
Do not load the scan
tray with the protective
base
GeneTitan 501025 The GeneTitan stain trays

Stain Trays are packaged in ziplock
bags to keep them free
of dust. The GeneTitan
stain trays are barcoded
and tray have separator
walls that are flush with
the frame of the stain
tray as shown by the
yellow line.

GeneTitan 202757 The GeneTitan scan and

Scan and stain tray covers are
Stain Tray provided to prevent any
cover evaporation of the
stains in stain trays and
the array holding buffer
in the scan tray
All stain and scan trays
must be placed in the
with the GeneTitan stain
tray cover.
All tray covers must be
de-ionized to remove
static electricity prior to
placing the cover on the
tray.
See the section
the anti-static
procedure.
GeneTitan Tray 501025 The GeneTitan stain

stain tray Cover 202757 trays must be placed in
shown with the GeneTitan
the stain instrument with the
tray cover GeneTitan stain tray
cover.
It is important to remove
the static electricity on
the cover and the stain
tray prior to loading the
tray into the GeneTitan
instrument.
See the section
the anti-static
procedure.
GeneTitan 500867 The GeneTitan

Hyb Tray hybridization trays are
packaged in white
pouches with the label
“HT Hybridization
Tray.”
After aliquoting the hyb
cocktail into the
hybridization tray, the
tray should be loaded
into the GeneTitan
instrument with the
barcode facing away
from the operator, i.e.,
Barcode should be on
the back side.
Proper Tray Alignment and Loading

Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC. Each
plate, cover and tray has one notched corner. The notched corner of plates, trays, covers and bases must
be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside
each GeneTitan MC drawer (Figure 3.6 and Figure 3.7 on page 34).
IMPORTANT: When running a multi-plate workflow, you must pay careful attention to the
software prompts that tell you which side of the drawer to place or remove a plate/tray.
TIP: Mark the notched corner of each plate, cover and tray with permanent marker to help
ensure proper alignment and loading onto the GeneTitan MC.
CAUTION: Take care not to damage the consumables or bend the blue base posts or scan tray
posts.
Figure 3.6 Proper alignment and loading of plates, covers and trays in the GeneTitan MC.
IMPORTANT: Remove the

plastic protective shipping
tray cover.
Notched corner of Array Plate aligned

with notched corner of blue base.
Tip
Mark the notched corner of each plate,
cover and tray with permanent marker to
help ensure proper alignment and loading.
Plates and trays must be seated in this

rectangular recess.
The notched corner of all plates, bases, and

covers and must be seated in this corner of
the drawer per the Tray Alignment guide.
Figure 3.7 Array Plate with protective blue base and the Hyb Tray aligned and properly loaded into drawer 6.
Array Plate with protective blue base Hyb Tray
IMPORTANT: When you install the consumables, ensure that the fingers are retracted (do
not put the consumables onto the drawer tab, or fingers - this indicates that the
instrument is not functioning correctly. See the Important note on on page 37 for an
image of the tabs.
Stain Trays and Covers
IMPORTANT: Always place the flat side of the cover against the Stain Tray.
Figure 3.8 Placement of covers on trays
Correct placement of cover on Stain Tray. Incorrect placement of cover on Stain Tray.
Labeling GeneTitan Hybridization and Reagent Trays

When preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument, you
will need to mark each tray in a way that identifies its contents.
IMPORTANT: It is critical that you write only on the proper locations of the proper sides of
Hyb and Stain Trays. Do NOT write in any other location, as this can interfere with sensors
inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper
placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also
mark the notched corner of the trays and lids.
Proper labeling for Hyb Trays and reagent trays is described in:
Labeling for Hyb Trays, below
Labeling for Stain Trays on page 36
Labeling for Hyb Trays
You may label the Hyb Tray on the front part of the short side of the tray, next to the notch at the left,
as shown in Figure 3.9. The proper section for labeling is closest to the notched corner, corresponding to
the A1 and B1 wells.
Figure 3.9 Labeling GeneTitan hyb trays
CAUTION: Writing on the wrong side of the Hyb tray may interfere with the operation of
sensors in the GeneTitan MC Instrument.
Labeling for Stain Trays

You may label the stain trays on the left side of the front of the tray as shown in Figure 3.10. The correct
side is closest to the notched corner, corresponding to the A1 through C1 wells.
IMPORTANT: Do not confuse hyb trays with stain trays.
Figure 3.10 Labeling GeneTitan stain tray
Loading Tray Consumables onto the GeneTitan MC

Loading, or installing, the trays and plates onto the GeneTitan MC Instrument is a simple procedure, but
there are certain precautions that you must take in order to ensure an error-free procedure. The following
figures show you how to do it.
Figure 3.11 Scan tray loaded in drawer 2.
Scan Tray with cover

loaded in drawer 2.
Do NOT load the protective black

base packaged with the Scan Tray.
IMPORTANT: When you load the plates, or trays, insert them under the tabs, or fingers, that
may protrude into the stage. Confirm that the tray is not resting on these fingers.
Figure 3.12 Stain 1 Tray and Ligation Tray loaded in drawer 3.
Stain 1 Tray and Ligation Tray

loaded in drawer 3
Figure 3.13 Stain 2 Tray and Fix Tray loaded in drawer 4.
Stain 2 Stain Tray and Stabilize

Tray loaded in drawer 4
Figure 3.14 Stain 1 Tray loaded in drawer 5.
Stain 1 Tray loaded in drawer 5.

Reagent Block Template

The Axiom Reagent Kit template fits precisely onto the top of the chilled reagent block, and is held in
place by the metal posts on the block (Figure 3.15). Using this template will help ensure the proper
placement of reagent tubes onto the block for each method.
Figure 3.15 Axiom reagent template for the

reagent block
Related Biomek FXP Target Prep Express Documentation

The following user manuals are installed at the same time as the Biomek FXP Target Prep Express
software (Start > All Programs > Beckman Coulter > Manuals). Refer to these for troubleshooting the
Biomek workstation.
Biomek® Liquid Handler User’s Manual, Beckman Coulter P/N 987834
Biomek® Software User’s Manual, Beckman Coulter P/N 987835
Stage 1 — DNA Amplification
IMPORTANT: Before proceeding to DNA Amplification, do the gDNA preparation described

in Chapter 2, Genomic DNA Preparation and Requirements on page 9.
NOTE: For this protocol, the term samples includes the positive control.
Duration
NOTE: A 22-24 hr incubation is required at the end of this stage.
Table 3.4 Time required for Stage 1 — DNA Amplification

Hands-on time ~ 30 min
Biomek FXP Target Prep Express ~ 19 min
Incubation 23 hr
Total time ~ 24 hr
Equipment and Labware Required
Table 3.5 Equipment and labware required

Quantity Item
1 Bench top cooler, chilled to –20 °C
As required Kimwipes®
1 Marker, fine point, permanent
1 Mini microcentrifuge (microfuge with microtube rotor)
1 Oven (must maintain a constant temperature of 30 °C for at least 23.5 hr with a temperature
accuracy of +/– 1 °C)
> 3 array plates per week — we recommend using the Binder ED 53
≤ 3 Array plates per week — OK to use the GeneChip Hybridization Oven or the Binder ED 53
1 Plate centrifuge
1 Vortex
Biomek Workstation Labware
1 box of each Barrier pipette tips:

Biomek Span P1000 (yellow)
Biomek AP96, P250 (aqua)
2 Plate, BIO-RAD hard shell PCR 96-well
2 Plate, deep well titer
1 Reagent block, chilled to 4 °C
2 Reservoir, quarter module (40 mL)
3 Reservoir, half module (75 mL)
Reagents Required
Table 3.6 Reagents required for DNA amplification

Axiom Reagent Kit Module
Axiom Denat Soln 10X
Axiom Neutral Soln 10X

Module 1, –20 °C
Axiom Amp Soln
Axiom Amp Enzyme
Axiom Water
1. Perform the Pre-Run Checklist
Check the Water and Waste Containers

To check the system water and waste containers:
1. If necessary, fill the system water container using distilled, deionized water (no need for ultra-pure
water).
2. If necessary, empty the system waste bottle.
Turn on the Biomek FXP Target Prep Express

To turn on the workstation:
1. Power on the workstation.
2. Ensure that all of the peripherals are powered on.
Watlow temperature controllers
Control the Static Peltier (Pelt_1) and the Shaking Peltier (SPelt_96); no additional power supply.
Thermal cycler:
BIO-RAD PTC-200 or Whatman Biometra TRobot 96 if present on the Biomek workstation deck.
Otherwise, a stand-alone thermal cycler can be used.
3. Launch the Biomek Software by double-clicking the Biomek Software icon on the desktop .
You can also open Start > All Programs > Beckman Coulter > Biomek Software.
Close the thermal cycler (on deck thermal cycler)

If your Biomek Workstation includes a PTC200 or a Biometra TRobot 96, the lid may remain open upon
startup. You must close the thermal cycler lid prior to homing the axes or starting a method.
The lid of the PTC-200 opens automatically when the device is powered up. While the lid of the TRobot
does not power up automatically, it is possible that the lid may be open from a previous run or at the
beginning of the next run.
CAUTION: Close the thermal cycler lid:

If it remains open after you have powered on the workstation.
If the lid is up before you home the axes or before you begin a method.
If not closed, the MC Pod may collide with the thermal cycler lid and damage the instrument.
NOTE: Refer to page 12 of the Setup Guide and User's manual for Biometra TRobot on the
Biomek FXp. After following the instructions to close the thermal cycler lid, proceed to Home
All Axes on page 44.
Closing the thermal cycler lid on the PTC-200

1. Open Instrument > Device Editor.
2. Open the Device drop-down menu and select PTC200Left.
3. Click Action Commands.

4. Select the following (Figure 3.16 on page 44):
A. In the Actions box, select Close.
B. In the Open/Close box, select Without plate.
IMPORTANT: It is critical that you select Without plate in the Open/Close box. If a plate
is present, remove it now.
5. Click Close Lid.

A Status window is displayed while the lid is being closed (Figure 3.17).
6. Click OK when the Command executed prompt is displayed (Figure 3.17).
7. Click Cancel; then click Close.
Figure 3.16 Closing the thermal cycler lid
Very Important to avoid

damaging the thermal cycler
Figure 3.17 Closing PTC Lid prompts.
Home All Axes

This procedure will home all axes and prime the fluidics lines.
To home all axes:
1. Open Instrument > Home All Axes.
2. Ensure all conditions in the Warning prompt Figure 3.18 (1.) are met, then click OK.
An icon instructing you to Stop and wait while the instrument homes is displayed (2.).
Figure 3.18 Homing all axes
1. 2.
3. When:
A. The Warning prompt in Figure 3.19 (A.) is displayed, confirm that no tips are loaded in the Span-
8 Pod, and click OK.
The lines for the Span-8 tips are primed and the next prompt shown in Figure 3.19 (B.) is
displayed.
B. When the intake (syringes and tubing) for the Span-8 tips is clear of bubbles, click OK.
Figure 3.19 Prompts displayed for priming the Span-8 Pod fluidic lines.
A. B.
2. Thaw and Prepare the Reagents and Sample Plate
Thaw and Prepare the Reagents and Sample Plate

To thaw and prepare the reagents:
1. Thaw the sample plate on the bench top at room temperature and spin.
NOTE: Do not place a frozen sample plate directly on the workstation deck.
2. Thaw the following reagents on the bench top at room temperature.

Axiom Amp Soln
Axiom Water
Leave the Axiom Amp Enzyme in the freezer until ready to use.
3. Vortex and spin all reagents (except Axiom Amp Enzyme), then place on ice.
Vortex the Axiom Amp Soln for 30 sec to thoroughly mix.
For the Axiom Amp Enzyme, just before placing on the deck gently flick the tube 3 times to mix
and spin.
4. Preheat the Oven to 30 °C.
We recommend using one of these ovens:
Binder FD53
Affymetrix GeneChip® Hybridization Oven (turn rotation on to 15 rpm)
4. Run the DNA Amplification Step

To run the DNA Amplification step:
1. Open Project > Open Project > Axiom Target Prep (Figure 3.20) and click OK.
Figure 3.20 Opening a project
2. Open File > Open to display the Open Method window (Figure 3.21).
Or click the Open Method icon
3. Select Axiom Target Prep and click OK.
Figure 3.21 Opening the Axiom Target Preparation method
The Axiom Target Prep

project folder must be
displayed in this menu.
4. At the top of the main window, click the Run button to open the Axiom™ Target Prep window
(Figure 3.22 – A.).
5. In the Axiom Target Preparation window (Figure 3.22 – B.):
A. Select your Array Plate format.
B. Select DNA Amplification.
C. Click OK.
The Deck Layout for DNA Amplification is displayed (Figure 3.23 on page 48).
Figure 3.22 Opening the Axiom Target Preparation methods window
B.
A. Run button
6. Place the labware and reagents on the deck as directed in the following figures and table:
Figure 3.23 on page 48 — deck layout
Table 3.7 on page 48 — labware and reagents
Figure 3.24 on page 49 — reagent block
IMPORTANT: Axiom Amp Enzyme — Immediately prior to placing on the reagent block,
gently flick the tube with your finger two to three times to mix; then spin. Do NOT vortex.
Figure 3.23 Deck layout window for the DNA Amplification method
Span-8 pod uses the pipette tips in

this position.
MC pod uses the pipette

tips in this position.
Important: No tips
should be loaded onto
the movable arms
referred to as the Span-8
(right) and the
Multichannel
(MC; left) pods.
Table 3.7 Labware and reagent locations on the deck for the DNA Amplification method
Position on Labware Reagent or Samples
Deck
TL1 Biomek AP96 – P250 Pipette Tips (aqua) —
P4 Beckman Deep Well Titer Plate gDNA samples
P5 BIO-RAD Hard Shell 96-well plate (any color) —
P6 BIO-RAD Hard Shell 96-well plate (any color)
SPelt96_1 Beckman Deep Well Titer Plate —
P10 Reservoirs in frame:

Quarter module (1)
Quarter module (2) 1 2 3

Half module (3)
Empty Empty Empty

Reservoir Reservoir Reservoir
Leave all reservoirs empty
Table 3.7 Labware and reagent locations on the deck for the DNA Amplification method
Position on Labware Reagent or Samples
Deck

Half module (1)
Half module (2) 1 2
Axiom Water Axiom

Pour Axiom Water into reservoir 1 Amp Soln
Pour Axiom Amp Soln into reservoir 2
Pelt_1 Reagent block, chilled to 4 °C See Figure 3.24.
P13 Biomek Span P1000 Pipette Tips (yellow) —
Figure 3.24 Placement of reagents on chilled reagent block for GeneTitan reagent tray preparation
IMPORTANT:
Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.
Press reagent tubes into the block to ensure that they are fully seated.
A1
Reagent block with template.

Only the positions used for this
method are shown.
Flick and spin the Amp Enzyme

before placing in block.
Axiom
A1 Denat
Soln 10X
Axiom
Diagram of reagent block Neutral
without template Soln 10X
Axiom
Amp
Enzyme
7. Check the deck layout to ensure that all labware and reagents are in the proper locations.
NOTE: If the physical deck does not match the Deck Layout and Confirmation window
exactly (Figure 3.23 on page 48), either modify the physical deck to match exactly or
choose Abort in the Deck Layout and Confirmation window.
8. Click OK.
The system flushes the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
9. At the prompt to repeat the Span-8 fluidics system flush:
Click No if no air bubbles are present.
Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
Figure 3.25 Flushing the Span-8 fluidics system to purge air bubbles.
Prompt for
Span-8 flush
The DNA Amplification step runs until the Amplification Master Mix has been added to the sample plate.
Once complete, the instructions and prompt shown in Figure 3.26 are displayed.
Follow the instructions for the sample plate (see also User Intervention below).
Follow the instructions for clearing the workstation deck.
Figure 3.26 Instructions for clearing the workstation deck
IMPORTANT:
Seal the sample plate before placing in the
oven.
Always discard the used multi-channel pipette
tips in position P3.
Always store the reagent block at 4 °C.
User Intervention
1. Remove the sample plate.
2. Blot the top of the plate with a Kimwipe to remove any droplets that may be present.
3. Tightly seal the plate.
4. Place in a preheated oven and incubate at 30 °C for 22 to 24 hr.
NOTE: If using a GeneChip® Hybridization Oven, place the plate on the bottom of the
oven. Plates do not rotate.
5. After 22 to 24 hr of incubation, do one of the following:

Proceed directly to Stage 2 — Fragmentation and Purification on page 53
Tightly seal and store the amplified samples at –20 °C.
Summary of DNA Amplification
MC Pod Activities Span-8 Pod Activities
3 min incubation on deck at

room temperature
Continued on next page.


Stage 2 — Fragmentation and Purification
NOTE: Purification is done by precipitation (4. Precipitation on page 61).
Duration
Table 3.8 Time required for Stage 2 — Fragmentation and Purification

Hands-on time ~ 25 min
~ 50 min if frozen DNA from Step 1
Biomek FXP Target Prep Express

Deactivation incubation — 40 min to deactivate the ~ 1:35 hr — 96 samples
amplification reaction
Fragmentation incubation — 30 min
Total time 2:00 to 2:25 hr
Table 3.9 Equipment, consumables and labware required

Quantity Item
1 Freezer, –20 °C
As required Kimwipes
1 Oven, preheated to 48 °C, with a temperature accuracy of +/– 1 °C
1 Plate centrifuge, 4 °C
1 Vortex (for plates and microtubes)
Beckman Labware

Biomek Span P250 (green)
3 Plate, BIO-RAD hard shell PCR 96-well
1 Plate, ABgene 96 Square Well Storage
3 Reservoir, quarter module
1 Reservoir, half module

Reagents Required
Table 3.10 Reagents required for the Fragmentation method

Reagent Module
Axiom Frag Enzyme (leave at –20 °C until ready to use)

Module 2
Axiom 10X Frag Buffer Box 1, –20 °C
Axiom Precip Soln 2
Axiom Frag Diluent

Module 2
Axiom Frag Rxn Stop Box 2, 2–8 °C
Axiom Precip Soln 1
User-supplied
Isopropanol, 99.5% 96 samples: 65 mL

The following actions are the same as described under 1. Perform the Pre-Run Checklist on page 42.
Refer back to this step for details. Some or all of these steps may not be required depending upon the
current state of the Biomek workstation.
To perform the pre-run checklist:
1. Power on the Biomek FXP Target Prep Express and all peripherals.
2. Check the water and waste containers; replenish or empty as required.
4. If applicable, close the thermal cycler lid (on page 42).
5. Home all axes (on page 44).
2. Thaw and Prepare the Samples and Reagents
Thaw and Prepare the Sample Plate

If the plate of amplified samples is frozen:
1. Place the deep well plate in a small water bath.
For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.
2. Leave the plate in the water bath for ~ 50 min until all wells have thawed.
3. Spin down at 1000 rpm for 30 sec.
4. To avoid cross-contamination of wells during vortexing:
A. Remove the seal and blot the top of the plate with a Kimwipe.
B. Tightly reseal the plate with a fresh seal.
5. Vortex the plate for 30 sec to thoroughly mix.
6. Spin at 1000 rpm for 30 sec.
Thaw and Prepare the Reagents

1. Thaw the following reagents on the bench top at room temperature.
Axiom 10X Frag Buffer
Axiom Precip Soln 2
2. Vortex and spin all reagents (except Axiom Frag Enzyme), then place on ice.
For the Axiom Frag Enzyme: Leave at -20 degrees C until ready to use. Just before placing on the
deck gently flick the tube 3 times to mix and spin
3. Run the Fragmentation Step

To open the Target Preparation Methods window:
1. Open Project > Open Project > Axiom Target Prep and click OK.
2. Click the Open Method icon, select Axiom Target Prep, and click OK.
3. Click the Run button.
4. Select the array plate format and Fragmentation, then click OK (Figure 3.27).
The deck layout for Fragmentation is displayed (Figure 3.28).
Figure 3.27
5. Setup the deck with the labware and reagents as shown in Figure 3.28 on page 56, Table 3.11 on
page 56, and Figure 3.29 on page 57.
IMPORTANT: Remove the seal from the sample plate before placing on the deck.
Figure 3.28 Deck layout for the Fragmentation method
Table 3.11 Labware and reagent locations on the deck for the Fragmentation
Position Labware Reagent or Samples
on Deck
P4 BIO-RAD Hard Shell 96-well plate —
P7 Beckman Deep Well Titer Plate Amplified gDNA
P8 ABgene 96 Square Well Storage Plate —

Quarter module (1)
Quarter module (2) 1 2

Axiom Axiom
Frag 10X Frag
Pour Axiom Frag Rxn Stop into reservoir 1 Rxn Buffer
Pour Axiom 10X Frag Buffer into reservoir 2 Stop

Half module (1)
Quarter module (2) 1 2

Axiom
Isopropanol Precip
Pour Isopropanol into reservoir 1 Sol 1
Pour Axiom Precip Soln 1 into reservoir 2
Table 3.11 Labware and reagent locations on the deck for the Fragmentation
on Deck
Pelt_1 Reagent block, chilled to 4 °C See Figure 3.29 on page 57.
P14 Biomek Span P250 Pipette Tips (green) —
IMPORTANT:
Always position the chilled reagent block with A1 in the upper left corner of the frame.
A1

method are shown.
Axiom Frag Enzyme:

Flick to mix 3X and spin
immediately prior to placing on
the chilled reagent block.
Axiom
A1 Frag
Enzyme
Axiom
Diagram of reagent block Frag
without template Diluent
Axiom
Precip
Soln 2
6. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.
NOTE: If the physical deck does not match the Deck Layout and Confirmation window
exactly (Figure 3.28 on page 56), either modify the physical deck to match exactly or
choose Abort in the Deck Layout and Confirmation window.
7. Click OK to continue.
The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air
bubbles.
The Fragmentation step begins. The sample plate is incubated at 65 °C to inactivate amplification. If you
selected Prompt for manual DNA quantitation in the default software settings, you will then be prompted
to remove an aliquot of each sample for a quantitation process control. The plate will remain at 40 °C
until the aliquots have been collected.
NOTE: Remain near the Biomek FXP Target Prep Express if you are going to remove
aliquots for quantitation. Avoid leaving the samples at 40 °C for a long period of time.
Summary of Fragmentation
Optional pause available for

manual DNA quantitation. See
Set the Biomek Software Default Incubate on deck:
Settings on page 17. 20 min at 65 °C
20 min at 40 °C
Incubate on deck
30 min at 40 °C


4. Precipitation
To freeze the samples:
1. Remove the Precipitation Plate from the deck.
2. Blot the top of the plate with a Kimwipe and seal tightly.
3. Place the plate in a –20 °C freezer overnight to precipitate.
4. Return to the Biomek workstation and clear the deck.
5. Centrifuge and Dry Pellets
CAUTION: During this step, handle the plate gently to avoid disturbing the pellets. Do not
bump or bang the plate.
To centrifuge and dry the pellets:

1. Turn the oven on and preheat to 48 °C.
Use an oven that can sustain a constant temperature of 48 °C and has a temperature accuracy of
+/– 1 °C (we recommend the Binder FD53). If processing three or fewer array plates, you can use a
GeneChip Hybridization Oven.
2. Centrifuge the plate at 3200 xg (or rcf) at 4 °C for 40 min.
.
WARNING: We strongly recommend that you use the Eppendorf 5810R at 3200 xg
(4000 rpm/) with:
Rotor A-4-81
Plate carrier DL 050 (rubber bottom)
If not using the Eppendorf 5810R with recommended rotor and plate carrier, centrifuge
the samples at 3200 xg (or rcf) at 4 °C.
Do not use the 5810R with the A-4-62 rotor and WO-15 plate carrier (hard bottom). Use of
this rotor and plate carrier may result in cracked plates, loss of sample, unbalanced
centrifugation, damage to the instrument and possible physical injury.
3. Immediately after the 40 min centrifugation period, empty the liquid from the plate as follows:
A. Remove the seal.
B. Invert the plate over a waste container and allow the liquid to drain.
C. While still inverted, gently press the plate on a pile of Kimwipes on a bench and leave it for 5 min.
4. Turn the plate right side up and place in an oven for 20 min at 48 °C to dry.
oven. Plates do not rotate.
5. Do one of the following:

Proceed directly to Stage 3 — Resuspension and Hybridization Preparation on page 62, even if
some droplets of liquid remain. Leave the sample plate at room temperature.
Tightly seal the plate and store at –20 °C.
Stage 3 — Resuspension and Hybridization Preparation
Duration
Resuspension and Hybridization Preparation
Table 3.12 Time required for resuspension

Hands-on time 15 min
Frozen pellet equilibration to room temperature 1.5 hr
Biomek FXP Target Prep Express 40 min
Total time 55 min to 2.6 hr

Quantity Item
1 Plate centrifuge, 4 °C
1 Vortex
Biomek FXP Target Prep Express Labware

Biomek Span P50 (pink)
Biomek Span P250 (green)
5 plates for BIO-RAD hard shell PCR 96-well

on-deck For on-deck thermal cycling or when using the PTC-0240/PTC-200 off-deck thermal cycler
cycling
OR:
6 plates for 4 BIO-RAD ( HSP-9631 ) Hard-Shell PCR 96-well Plate and

off-deck 1 HSS-9601 Hard-Shell Full-Height 96-Well Semi- Skirted PCR Plate
cycling 1 Plate, Costar Brand Serocluster round bottom plate
For off-deck thermal cycling using the ABI 9700 or ABI 2720 thermal cycler
Note: The HSS-9601 plate stacked on the Costar brand serocluster round-bottom plate should
only be used on position P2 on the Biomek FXp deck. See Figure 3.35 on page 75.
1 Plate, OD

Quantity Item
1 Reservoir, 19 mL (quarter module, divided)
3 Reservoir, 40 mL (quarter module)
Reagents Required
Table 3.14 Reagents required for resuspension and hybridization

Reagent Module
Axiom Hyb Buffer Module 2

Box 1, –20 °C
Axiom Hyb Soln 1
Axiom Resusp Buffer Module 2

Box 2, 2–8 °C
Axiom Hyb Soln 2
Other Reagents Required
Nuclease-Free Water, ultrapure MB grade To fill line of divided reservoir

(USB, P/N 71786; for OD and gel plate preparation)
TrackIt Gel Loading Buffer, diluted 96-array plate: 8 mL

(see Appendix A, Fragmentation Quality Control Gel Protocol on
page 223 for dilution instructions.)
1. Preparing Frozen Pellets and Axiom Resusp Buffer
IMPORTANT: The pellets and the resuspension buffer must be at room temperature before
proceeding with this step.
Allow the pellets and the Axiom Resusp Buffer to equilibrate to room temperature for 1.5 hr prior to
starting this step.

4. If applicable, close the thermal cycler lid (on page 42).
3. Thaw and Prepare the Reagents
Thaw and Prepare the Reagents

1. Thaw Axiom Hyb Soln 1 on the bench top at room temperature.
2. Vortex the Axiom Resusp Buffer and the Axiom Hyb Buffer.
3. Vortex and spin Axiom Hyb Soln 1 and Axiom Hyb Soln 2.
4. Run the Resuspension and Hybridization Preparation Step
NOTE: We strongly recommend that you run two quality process controls during this step:
A gel to verify successful fragmentation
An OD quantitation of each resuspended sample
The Biomek FXP Target Prep Express can be set to prepare fragmentation and OD plates that
are ready for processing. These process controls must be selected as a run preference prior to
starting a run. See Set the Biomek Software Default Settings on page 17 for instructions.
To run the Resuspension and Hybridization Preparation step:

1. Open Project > Open Project > Axiom Target Prep and click OK (Figure 3.30).
4. Select your array plate format and Resuspension and Hybridization Preparation.
The deck layout for this method is displayed (Figure 3.31 on page 65).
Figure 3.30 Selecting the Resuspension and

Hybridization Preparation method
5. Prepare the deck as shown in Figure 3.31, Table 3.15 and Figure 3.32 on page 67.
Label the BIO-RAD plates placed on the deck in positions P2 and P11. For example:
P2 — Hyb Ready + <sample description>
P11 — Gel QC
NOTE: Verify the appropriate plastic consumables are being used on the deck for the Hyb
Reaction Plate when using the ABI 9700 or ABI 2720 thermal cycle.
Figure 3.31 Deck layout for the Resuspension and Hybridization Preparation method
If Prepare plates for gel QC and OD

after resuspension is selected in your
run preferences, the following
labware is required on the deck:
Multichannel P50 pipette tips
Dilution QC plate
Gel QC plate
OD QC plate (do NOT touch
bottom of plate)
If not selected, these plates will not
appear in the deck layout.
See Set the Biomek Software
Default Settings on page 17 for
more information.
Table 3.15 Labware and reagent locations on the deck for Resuspension and Hybridization Preparation
on Deck
P1 ABgene 96 Square Well Storage plate Pelleted samples
P2 BIO-RAD Hard Shell 96-well plate

(Hyb Reaction Plate).
Bio-Rad Hard Shell 96-well plate (HSP 9631) for
on-deck or off-deck thermal cycling using the

TRobot 96, PTC-200 or PTC 0240.
Bio-Rad Hard-Shell Full-Height 96-Well Semi-
Skirted PCR Plate (HSS 9601) stacked on a

Costar Round Bottom plate for off-deck
thermal cycling with the ABI 9700 or 2720
thermal cycler
Table 3.15 Labware and reagent locations on the deck for Resuspension and Hybridization Preparation (Continued)
on Deck
Warning: When using the ABI 9700 or the ABI 2720 off-deck thermal cycler for denaturing the hyb-ready
plate, the Bio-Rad HSS 9601 Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate must be stacked on a
CoStar Brand Serocluster Round Bottom plate (29442-392 from VWR International or EK-680568 from E&K
Scientific) as shown in Table 3.2 on page 22.
The HSS9601 and HSP9631 PCR plates are not interchangeable on the Biomek FXp deck.
P3 Biomek Span P50 Pipette Tip (pink) —

Quarter module (1)
4
Quarter module (2) 1 2 3 Gel Load
Quarter module (3)
Buffer
Axiom Axiom
Quarter module, divided (4 and 5) Resusp Hyb Empty
Buffer Buffer Reservoir 5
Water
Pour Axiom Resus Buffer into reservoir 1
Pour Axiom Hyb Buffer into reservoir 2
Leave reservoir 3 empty
Pour diluted gel loading buffer into reservoir 4:
– 8 mL for 96 samples
Pour nuclease-free water into reservoir 5 to fill
line
P11 BIO-RAD Hard Shell 96-well plate (for Gel QC) —
P12 OD plate, 96-well UV —
Pelt_1 Reagent block, chilled to 4 °C See Figure 3.32 on page 67.

IMPORTANT:
A1

method are shown.
Axiom
A1 Hyb
Soln 1
Diagram of reagent block Axiom

without template Hyb
Soln 2
NOTE: If the physical deck does not match the Deck Layout window exactly (Figure 3.31
on page 65), either modify the physical deck to match exactly or choose Abort in the Deck
Layout window.
7. Click OK to continue the method.

bubbles.
9. Run the fragmentation gel and OD quantitation process controls.
For instructions and guidelines on assessing gel and OD results, see:
Appendix A, Fragmentation Quality Control Gel Protocol on page 223
Appendix B, Sample Quantitation after Resuspension on page 225

If the GeneTitan MC Instrument is free, and if the gel and OD quantitation results are good, you
can proceed directly to Stage 4 — Preparation for the GeneTitan MC Instrument on page 72.
If the GeneTitan MC Instrument is not free, then follow the instructions shown in Figure 3.33.
Tightly seal the Hyb Rxn plate and store at –20 °C.
Figure 3.33 Instructions to follow if the GeneTitan MC Instrument is not free

Summary of Resuspension and Hybridization Preparation



Stage 4 — Preparation for the GeneTitan MC Instrument
About Stage 4
You will proceed to Stage 4 in one of two ways:
Directly from Stage 3 without interruption.
With samples that were stored at –20 °C after Stage 3.
This stage, Preparation for GeneTitan, can include any combination of the options shown in Figure 3.36
on page 76 and Figure 3.37 on page 82. The first two options complete target preparation on the Biomek
FXP Target Prep Express.
The options are:
Option 1
Denature samples - the Hyb Rxn plate is placed on the thermal cycler and the samples are denatured.
At this step, you must also select Transfer denatured samples to Hyb Tray. After the denature program
has completed, the block will hold temperature until the user has dismissed the prompt, indicating that
they are ready to continue the method to transfer the samples to the hyb tray and then carry it to the
GeneTitan. Do not leave samples on the thermal cycler for a long period of time.
Option 2
Transfer denatured samples to Hyb Tray - the denatured samples are transferred from the Hyb Rxn
plate to the Hyb Tray, and are ready to load onto the GeneTitan MC Instrument.
NOTE: When using an ABI 9700 or the ABI 2720 thermal cycler for off-deck denaturation, the
Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (hyb reaction plate) must be
stacked on a Costar brand Serocluster Round Bottom plate from Corning (Corning Mfg P/N
3795)
Option 3
Prepare GeneTitan® reagent plates (option 3) - the solutions required for the fluidics stage of array
processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (three
stain trays, one ligation tray, one fix tray and one scan tray with Holding Buffer).
When performing a 1 plate workflow, select two options (option 1 and option 2).
Denature samples and Transfer denatured samples to hyb tray when you are preparing the samples to
begin hybridization in the GeneTitan.
- or select one option (option 3)-
Prepare GeneTitan reagent plates to prepare reagents for loading onto the GeneTitan the following
day when the plate is finished with the hybridization period and about to begin GeneTitan fluidics
processing.
Options for performing a multi-plate workflow

When performing a multi-plate workflow (see Chapter 6, Automated Target Preparation for Processing
Eight Axiom Array Plates Per Week on page 183 for a description of the 8 plate workflow), the need to
carry out preparation of reagents for one plate while simultaneously hybridizing a second plate will arise.
In this case, all three options in the Biomek FXp method can be carried out concurrently. Select all three
options when using an on-deck thermal cycler.
Denature samples and Transfer denatured samples to hyb tray will prepare samples for the new plate
going into the GeneTitan to begin hybridization.
-and-
Prepare GeneTitan reagent plates will prepare reagents for the plate that is already in the GeneTitan
hyb oven and is ready to go into the Wash, Ligation, Stain, and Scan phases of GeneTitan array
processing.
NOTE: In the 8 plate workflow, all three options will only be selected for middle days of the
workflow. On the first day of the workflow, only the Denature samples and Transfer
denatured samples to hyb tray options will be used. On the last day of the workflow, only
the Prepare GeneTitan reagent plates option will be used.
NOTE: Note: When using an off-deck thermal cycler, avoid letting the samples sit a room
temperature for an extended period of time after denaturation. Do not begin denaturation
at the same time as the GeneTitan reagent preparation.
Off-deck thermal cycler option

The steps for performing a simultaneous preparation of GeneTitan reagent plates and hybridization of a
second plate required in the course of a multi-plate workflow are modified slightly when the off-deck
thermal cycler option is selected. The reagent trays to be loaded into the GeneTitan MC instrument are
prepared in an initial run of the method.
Denaturation of the hyb ready samples in the off-deck thermal cycler begins mid way through the
GeneTitan reagent prep on the Biomek deck.
After loading the reagent trays into the GeneTitan MC instrument, you must perform a second run of the
Biomek method to transfer the denatured samples to the hyb tray for loading into the GeneTitan MC
instrument.
The modified steps are:
1. Select Preparation for GeneTitan step with only the Prepare GeneTitan reagent plates box checked,
click OK.
2. Prepare deck as shown in Figure 3.34.
Figure 3.34 Deck Layout for Off-deck thermal cycler option
3. Click OK.
4. Wait 18 minutes, then begin denaturation of the hyb ready samples using the off-deck thermal cycler.
5. Upon completion of the GeneTitan reagent plate preparation, load reagent plates and scan tray into
the GeneTitan MC instrument
6. Once the reagents are loaded into the GeneTitan MC instrument and the denaturation method on the
thermal cycler is complete, retrieve the denatured hyb ready samples from the thermal cycler
7. Return to Biomek FXp and begin a new method, select Preparation for GeneTitan step with only the
Transfer denatured samples to hyb tray box checked, click OK.
8. Prepare deck as shown in Figure 3.35 (note that a spacer must be used with HSS-9601 plates for ABI
thermal cyclers).
Figure 3.35 Deck Layout for Off-deck thermal cycler option and position P2
The Hyb Reaction Plate in deck position P2 must

be one of the following:
Bio-Rad Hard Shell 96-well plate (HSP 9631) for

on-deck or off-deck thermal cycling using the TRobot
96, PTC-200 or PTC 0240
OR
Bio-Rad Hard-Shell Full-Height 96-Well Semi- Skirted
PCR Plate(HSS 9601) stacked on a Costar Round
Bottom plate for off-deck thermal cycling with the
ABI 9700 or 2720 thermal cycler
9. Click OK.
10. After the denatured samples have been transferred to the GeneTitan hyb tray, load hyb tray into
GeneTitan
If a plate is already in the hybridization oven

For the plate that is already in the GeneTitan hyb oven and that is ready to go into Ligation, Wash-Stain
and Scan, select option 3, Prepare GeneTitan reagent plates. The solutions required for the fluidics stage
of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays
(three stain trays, one ligation tray, one fix tray and one scan tray with Holding Buffer).
IMPORTANT: The reagent plates prepared in the third sub-step, Prepare GeneTitan® reagent
plates:
Are NOT for use with the Hyb Tray currently being prepared on the Biomek workstation.
Are for the continued processing of an Axiom Array Plate that is:
– already on the GeneTitan MC Instrument.
– has completed the hybridization stage.
– is ready for transfer to the fluidics area.
The reagent plates for the fluidics stage on the GeneTitan MC Instrument CANNOT be
prepared in advance. Do not prepare these plates if there is no array plate ready for the
fluidics stage. Once prepared, these plates must be loaded onto the instrument as soon as
possible and cannot be stored.
Figure 3.36 Software setup for Preparation for GeneTitan
You can run one step, or a combination of steps.

Duration
Denaturation
Table 3.16 Time required to denature samples

Hands-on time: ~ 3 min
Biomek FXP Target Prep Express 23 min
Total time: ~ 26 min
Denatured Sample Transfer to Hyb Tray
Table 3.17 Time required to transfer denatured samples to the Hyb Tray
Hands-on time 2 min
Total time ~ 4 min
Preparation of GeneTitan Reagent Trays
Table 3.18 Time required to prepare reagent trays for the GeneTitan MC Instrument
Hands-on time ~15 min
Prepare reagents ~ 30 min
Total time ~ 75 min
NOTE: When you select Denaturation and Preparation of the GeneTitan Reagent Trays, the
on-deck processes run concurrently.
Equipment and Consumables Required
Denature Samples
Table 3.19 Equipment required for denaturing sample.

If denaturing samples: Item Quantity
On the Biomek workstation Lid, arched metal 1
Transfer to Hyb Tray
Table 3.20 Consumables required for transferring denatured samples to a Hyb Tray
Item Quantity
Hyb Tray 1
(from an Axiom Genome-Wide CEU 1 Array Plate Kit or the
GeneTitan Consumable Kit, P/N 901606)
Table 3.20 Consumables required for transferring denatured samples to a Hyb Tray
Item Quantity
Pipette tips, Biomek AP96 – P250 (aqua) 1 full box
Costar brand Serocluster Round Bottom plate from Corning 1

Corning Mfg PN 3795) to be used for stacking under the
HSS-9601 semi-skirted (hyb reaction plate) plate used with
the ABI 9700 and ABI 2720 thermal cyclers
Prepare GeneTitan Reagent Plates
Table 3.21 Consumables and other equipment required

Item Quantity
Axiom Genome-Wide CEU 1 Array Plate Kit or the 1 Scan Tray with cover
GeneTitan Consumable Kit: and protective base
Scan Tray with cover 5 Stain Trays
Stain Tray 5 Covers
Cover for Stain Tray
Pipette tips, Biomek AP96 – P250 (aqua) 1 full box
Pipette tips, Biomek Span P1000 (yellow) 1 full box
Pipette tips, Biomek Span P250 (green) 1 full box
Reagent block, chilled to 4 °C 1
Reservoirs, quarter module divided 3
Reservoirs, quarter module 3
Tube rack, 24 position with insert 1

(room temperature rack)
Zerostat AntiStatic Gun 1
NOTE: See Table 3.3 on page 28 for GeneTitan MC Consumable part numbers.

4. If open, close the thermal cycler lid (on page 42).
2. Prepare the Reagents for GeneTitan Reagent Plate Preparation
IMPORTANT: Ligation Buffer and Ligation Solution 2 require approximately 30 to 40 min to

thaw on the bench top at room temperature.
Reagents Required
Table 3.22 Reagents required for GeneTitan MC Instrument reagent plate preparation.
Module Reagent Thaw on Bench Place on Ice Place on Bench
Top, Then Place Top at Room
on Ice Temperature
Axiom Wash Buffer A

Module 3
Room Axiom Wash Buffer B
Temperature
Axiom Water
Axiom Ligate Buffer

for 30 min
Axiom Ligate Enzyme Keep at –20 °C

Module 4 until ready to use
Box 1 of 2
–20 °C Axiom Ligate Soln 1
Axiom Probe Mix 1
Axiom Stain Buffer
Axiom Stabilize Soln
Axiom Ligate Soln 2

for 30 to 40 min
Axiom Probe Mix 2*
Axiom Wash A
Module 4 for 30 min
Box 2 of 2
2 to 8 °C Axiom Stain 1-A*
Axiom Stain 1-B*
Axiom Stain 2-A*
Axiom Stain 2-B*
Axiom Stabilize Diluent
Axiom Water
Axiom Hold Buffer*
* These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.
Preparing Axiom Wash A

During storage of the Axiom Wash A from Module 4 at 4 °C, precipitation in the form of clear crystals
can sometimes occur. Therefore, follow the procedure below to ensure that any precipitate is returned to
solution prior to use.
NOTE: The presence of some precipitate is OK and will not adversely impact assay
performance. Follow the instructions below to resuspend any precipitate before use.
To prepare the Axiom Wash A:

1. Vortex the bottle for 30 sec.
2. Place on the bench top at room temperature for 30 min.
3. Examine the reagent for precipitate (look into the top of the bottle).
4. If precipitate is still present, vortex again for 30 sec.
5. Pour Axiom Wash A into the appropriate reagent reservoir and leave on the bench top.
Preparing Axiom Ligate Buffer

White precipitate is sometimes observed when the Axiom Ligate Buffer is thawed. Follow this procedure
to ensure that any precipitate is returned to solution prior to use.
NOTE: The presence of some precipitate is OK and will not adversely impact assay
performance. Follow the instructions below to resuspend any precipitate before use.
To prepare the Axiom Ligate Buffer:

1. Place on the bench top at room temperature for 30 min.
2. Examine the buffer for precipitate (look into the top of the bottle).
3. If precipitate is present, vortex the bottle for 30 sec.
4. Re-examine the buffer for precipitate.
5. If precipitate is still present, warm the bottle with your hands and vortex again for 30 sec.
6. Pour the Axiom Ligate Buffer into the appropriate reagent reservoir and leave on the bench top.
Prepare the Remaining Reagents

To prepare the remaining reagents for GeneTitan MC Instrument plate preparation:
1. Leave the Axiom Ligate Enzyme at –20 °C until ready to use.
2. Thaw the following reagents from Module 4, box 1 of 2 on the bench top at room temperature, then
vortex, spin and place on ice:
Axiom Ligate Soln 1
Axiom Probe Mix 1
Axiom Stain Buffer
3. Prepare the remaining reagents from Module 4, box 2 of 2 as follows:

A. Gently flick each tube 2 to 3 times to mix, then spin.
B. Place reagents on ice, except for the Axiom Ligate Soln 2 and Axiom Water— leave these reagents
on the bench top at room temperature.
3. Prepare the Sample Plate if Stored at –20 °C and the Array Plate
To prepare samples that were stored at –20 °C:
1. Vortex briefly; then spin at 1000 rpm for 30 sec.
2. Place on ice.
To prepare the Array Plate:

1. Remove the array plate box from the 4 °C refrigerator where it is stored.
2. Open the box and remove the pouch containing the array plate and protective base.
WARNING: Do not remove the array plate from the protective base or touch the surface
of any arrays.
3. Leave the array plate in the pouch, unopened but placed on the bench for a minimum of 25 minutes
before opening and loading on the GeneTitan MC to allow the plate to come to room temperature.
4. Run the Preparation for GeneTitan® Step

To run the Preparation for GeneTitan step:
1. Open Project > Open Project > Axiom Target Prep and click OK.
4. Select your array plate format.
5. Select Preparation for GeneTitan® and the sub-steps that you wish to run; then click OK.
Based upon your choices, the appropriate deck layout is displayed (Figure 3.38).
6. Deionize the GeneTitan stain trays. Refer to the section Deionization Procedure for GeneTitan Trays
and Covers on page 241 for the deionization procedure.
IMPORTANT: It is important to deionize the GeneTitan Instrument trays to remove any static
electricity on the trays. Static attraction by the trays may prevent the tray cover from being
lifted up by the instrument.
7. Prepare the deck as shown in:

Figure 3.38
Table 3.23 and Figure 3.41 on page 86
Figure 3.42 on page 86
NOTE: Prior to placing the arched metal lid on the deck, be sure to clean the attached
Microseal P pad with 70% ethanol and dry it. Refer to the package insert for this product
for further information on cleaning and replacement.
Figure 3.37 Software setup for Preparation for GeneTitan
Select Denature Samples and Transfer denatured samples

to hyb tray
if you are preparing to load the array plate into the
GeneTitan Instrument for Hybridization.
Select Prepare GeneTitan Reagent Plates
if you are preparing to load the GeneTitan Instrument for
Wash-Stain and Imaging.
Select Denature Samples and Transfer denatured samples
to hyb tray and Prepare GeneTitan Reagent Plates
if you are running an 8 plate workflow then you will need
to select all three check boxes in some instances in
preparation for the plate that will go into the GeneTitan
Instrument for Hybridization.
Select Prepare GeneTitan Reagent Plates for the array plate
that has completed hybridization and is ready to be processed
in the GeneTitan Instrument for Ligation- Wash-Stain and
Imaging.
You can run each step individually, or any

combination of steps. If you do not select Prepare
GeneTitan reagent plates, the deck layout will be
different than the one shown in Figure 3.38.
Figure 3.38 Deck layout for Preparation for GeneTitan — denature, transfer to Hyb Tray, and GeneTitan plate preparation
IMPORTANT: Destatic the bottom of the Hyb Tray, Stain Trays, and the protective base of the Scan Tray prior to
placing on the deck. See the section Deionization Procedure for GeneTitan Trays and Covers on page 241.
Included in deck layout if Prepare GeneTitan®

reagent plates is selected.
The deck layout displayed is based

upon the selections you make for
this step.
Included in deck layout if:

Denature samples
Transfer denatured
samples to Hyb Tray are

selected.
Table 3.23 Labware and reagent locations on the deck for GeneTitan reagent preparation
on Deck
If Denature samples and Transfer denatured samples to Hyb Tray is selected, (no reagent tray preparation), only
the labware listed below is required:
P1 Lid, arched metal —

Clean before use (70% ethanol).
P2 Hyb Reaction Plate++ Hyb-ready samples
P3 Hyb Tray* —
If Prepare GeneTitan® reagent plates, the labware listed below is required:
P4 Scan Tray on protective base* —
P5 Fix* —
P6 Stain 1* (first of two trays with Stain 1) —
P7 Stain 1* (second of two trays with Stain 1) —
P8 Stain 2* —
P9 Lig* —
P10 Tube block with one insert, room temperature See Figure 3.42 on page 86.

1 3 5
Quarter module, divided (1 and 2) Axiom
Quarter module, divided (3 and 4)
Axiom Ligate
Empty
Quarter module, divided (5 and 6)
Water Buffer
2 4 6
Pour Axiom Water into reservoir 1 Empty Empty Empty
Pour Axiom Ligation Buffer into reservoir 5

Quarter module (1)
1 2 3
Quarter module (2)
Quarter module (3)

Axiom Axiom
Hold Wash A Empty
Buffer Reservoir
Pour Axiom Hold Buffer into reservoir 1
Pour Axiom Wash A into reservoir 2
Leave reservoir 3 empty
Pelt_1 Reagent block, chilled to 4 °C See Figure 3.41 on page 86

++Bio-Rad Hard Shell 96-well plate (HSP 9631) for on-deck or off-deck thermal cycling using the TRobot 96, PTC-200
or PTC 0240. Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (HSS 9601) stacked on a Costar Round
Bottom plate for off-deck thermal cycling with the ABI 9700 or 2720 thermal cycler
*These trays are included in Axiom Genome-Wide and Custom myDesign™ Array Plate Kits.
Label each of these stain trays as described above as described in Labeling GeneTitan Hybridization and
Reagent Trays. For example, label the Stain tray to be placed in P9 with the word Lig. This tray will contain the
Ligation master mix.

hyb and stain trays. Do NOT write in any other location, as this can interfere with sensors

as shown in Figure 3.39. The proper section for labeling is closest to the notched corner, corresponding
to the A1 and B1 wells.
Figure 3.39 Labeling GeneTitan Hyb Trays
Writing on the wrong area of the Hyb tray may interfere with the operation of sensors in the GeneTitan
MC.
Figure 3.40 Labeling GeneTitan stain trays

IMPORTANT:
A1

method are shown.
Axiom Axiom Axiom

A1 Stabilize Stain 1- Ligate
Diluent A Soln 1
Diagram of reagent block Axiom Axiom Axiom

without template Stabilize Stain 1-B Probe
Soln Mix 1
Axiom Axiom
Stain 2- Probe
A Mix 2
Axiom Axiom Axiom

Stain Stain 2-B Ligate
Buffer Enzyme
Figure 3.42 Placement of reagents on room temperature tube block
Axiom
A1 Ligation
Soln 2
NOTE: If the physical deck does not match the Deck Layout window exactly (Figure 3.38
on page 82), either modify the physical deck to match exactly or choose Abort in the Deck
Layout window.
9. Click OK to continue the step.

bubbles.
If the “Denature Samples” option is selected, the Biomek FXP Target Prep Express places the samples
onto the thermal cycler and runs the denaturation program. If Prepare GeneTitan reagent plates is also
selected, reagent plate preparation for the GeneTitan MC Instrument starts while the samples are being
denatured.
6. Prepare the GeneTitan MC Instrument

While your samples are being denatured and/or the Biomek FXP Target Prep Express is preparing reagent
plates, ensure that the GeneTitan MC Instrument is ready for use.
One or more of the following steps may need to be performed:
1. Launch AGCC and select AGCC GeneTitan Control.
2. Upload your sample registration file now.
If you do not upload your samples before scanning the array plate barcode, the software will assign
names to your samples.
3. Select the System Setup tab (Figure 3.43).
4. Configure the software as follows:
A. Setup Option: Hyb-Wash-Scan
Other options available are described under Setup Options for Array Plate Processing on
page 154.
B. Click Next.
C. Plate Information:
Barcode: Scan or manually enter the Axiom Array Plate barcode and click Next.
D. Protocol Name: Select the protocol name and click Next.
5. Fill the Wash A, Wash B and Rinse bottles.
6. Empty the Waste bottle.
After preparing the GeneTitan MC Instrument, perform steps 7a, 7b or 7c as appropriate for your
workflow:
Go to Step 7a: if you are denaturing the samples and transferring them to the hyb tray.
Go to Step 7b: if you are preparing reagents for the Ligation-Wash-Stain
Go To Step 7c: if you are performing a 2-8 plate workflow, and you are denaturing samples for the
2nd plate and are preparing ligation reagents for the 1st plate. The samples for the 2nd HT array plate
will be denatured and transferred into the Hyb tray. The Hyb tray will be placed in the GeneTitan
with the array plate in preparation for hybridization. The robot will also prepare reagents for
Ligation-Wash-Stain for the 1st array plate that was placed in the GeneTitan hyb oven 24 hours ago.
Figure 3.43 Setup options for processing array plates
Step 7a. Complete Stage 4 — Preparation for GeneTitan - Hybridization Trays

If you selected Denature samples and Transfer denatured samples to Hyb tray in Step 4 (See Step 4.
Run the Preparation for GeneTitan® Step on page 81 and Figure 3.37 on page 82), use the following
instructions:
Figure 3.44 Prompt indicating denaturation is complete.
IMPORTANT: Immediately load the Array Plate and Hyb Tray into the GeneTitan MC
Instrument.
1. Once the GeneTitan MC Instrument is ready, return to the Biomek FXP Target Prep Express click OK
(prompt shown in Figure 3.44 above).
The denatured samples are then taken off the thermal cycler and are transferred to the Hyb Tray.
Ensure that there are no air bubbles present in the hyb tray. Puncture any air bubbles that you see
using a pipette tip.

2. Transfer the Hyb Tray to the GeneTitan MC Instrument and load. Refer to the section Axiom™
Genotyping Assay: Array Processing with the GeneTitan® MC Instrument on page 149 for the proper
way of loading the array plate and the hyb tray.
3. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 3.45).
Always discard the used multi-channel pipette tips in position P9.
Clean the Microseal P Pad by wiping with 70% EtOH and dry.
Refer to the package insert for this product for further information on cleaning and replacement.
Figure 3.45 Instructions displayed at the end of Step 4.
Step 7b. Complete Stage 4 - Preparation for GeneTitan - Reagent Trays

If you selected Prepare GeneTitan Reagent Plates in Step 4 (See Step 4. Run the Preparation for
GeneTitan® Step on page 81 and Figure 3.37 on page 82), use the following instructions:
1. Once the prompt shown in Figure 3.45 appears, remove the reagent plates and Scan Tray with Hold
Buffer from the deck and cover with the appropriate lids.
2. Examine each tray to ensure that:
All of the wells as appropriate (96) have been filled. If any wells do not contain reagents, then
manually add reagents to these wells.
There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.
j
IMPORTANT: Immediately load the reagent plates onto the GeneTitan MC Instrument. Do
not leave denatured samples or reagent plates at room temperature for any length of time.
3. Transfer the reagent plates, Scan Tray to the GeneTitan MC Instrument and load. Refer to the section
Stage 3 — Ligate, Wash, Stain and Scan on page 172 to continue the process on the GeneTitan MC
instrument
4. Return to the Biomek FXP Target Prep Express and clear the deck (See Figure 3.43 on page 88).
7c. Complete Stage 4 — Preparation for GeneTitan - Multiple plate workflow

If you selected all three options in Step 4 (See Step 4. Run the Preparation for GeneTitan® Step on
page 81 and Figure 3.37 on page 82)) and are preparing hyb tray and reagent trays in a 2+ plate workflow,
use he following instructions:
The Hyb tray is prepared for a new HT array plate that will be loaded into the GeneTitan. The reagent
trays are prepared for the HT array plate that is in the hybridization oven in the GeneTitan MC and is
ready to move to the next stage of the process - Ligation, Wash-Stain and Scan.
NOTE: Note: When using an of-deck thermal cycler please refer to the instructions for the Off-
deck thermal cycler option on page 73.
1. Once the reagent plates are prepared and sample denaturation is complete, the prompt in Figure 3.44
on page 88 is displayed (do not click OK yet). Remove the reagent plates and Scan Tray with Hold
Buffer from the deck and cover with the appropriate lids.
2. Examine each tray to ensure that:
All of the wells as appropriate (96) have been filled. If any wells do not contain reagents, then
manually add reagents to these wells.
There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.
3. Transfer the reagent plates, Scan Tray and Array Plate to the GeneTitan MC Instrument and load.
IMPORTANT: Immediately load the reagent plates and the Hyb Tray onto the GeneTitan
MC Instrument. Then load the Array Plate and Hyb Tray. Do not leave denatured samples
or reagent plates at room temperature for any length of time.
4. Return to the Biomek FXP Target Prep Express click OK at the prompt shown in Figure 3.44 on
page 88.
The denatured samples are then taken off the thermal cycler and are transferred to the Hyb Tray.
5. Transfer the Hyb Tray to the GeneTitan MC Instrument and load. Refer to the section Axiom™
Genotyping Assay: Array Processing with the GeneTitan® MC Instrument on page 149 for the proper
way of loading.
6. Transfer the reagent plates, Scan Tray to the GeneTitan MC Instrument and load. Refer to the section
Axiom™ Genotyping Assay: Array Processing with the GeneTitan® MC Instrument on page 149 to
continue the process on the GeneTitan MC Instrument
7. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 3.45 on page 89).
8.Summary of Preparation for GeneTitan MC Instrument
Continued on next page Continued on next page




4
Axiom Genotyping Assay:

Manual Target Preparation
Manual target preparation for the Affymetrix Axiom Genome-Wide assay enables you to perform target
preparation to process 96 samples at a time without the use of automation equipment.
NOTE: Array handling and processing protocols still require the use of a GeneTitan MC, as
described in Chapter 5, Axiom™ Genotyping Assay: Array Processing with the GeneTitan®
MC Instrument on page 149.
IMPORTANT: Read all the instructions in Before You Start, below, before performing manual
target preparation.
A list of all equipment and resources required for the Axiom Assay with manual target preparation is in
the Axiom Site Prep Guide P/N 702858.
The protocol for manual target preparation is presented in the following sections:
Before You Start, below
Stage 1 — DNA Amplification on page 107
Stage 2 — Fragmentation and Precipitation on page 115
Stage 3 — Drying, Resuspension and QC on page 122
Stage 4 — Denaturation and Hybridization on page 128
Stage 5 — Manually Preparing Ligation, Staining, and Stabilization Reagent Trays for the GeneTitan
MC Instrument on page 134
IMPORTANT: Before proceeding to DNA Amplification, perform the gDNA preparation

described in Chapter 2, Genomic DNA Preparation and Requirements on page 9.
Using the manual target preparation protocol, a single operator can process three sample and array plates
a week during a forty-hour work week for a total of 288 samples. See Chapter 8, Manual Target
Preparation for Processing Three Axiom Array Plates per Week on page 209 for more information.
Before You Start

This section provides information on procedures that are performed multiple times during manual target
preparation, and on steps that are critical to the success of the manual target preparation. It is essential
that you familiarize yourself with the information in this section prior to running the manual target
preparation for Axiom™ Genotyping Assay.
Requirements and Recommendations

This section describes requirements and recommendations for facilities and equipment needed to perform
the Axiom Genotyping Assay with manual target preparation.
Room Temperature
When referred to in the Axiom Genome-Wide Assay, room temperature is 18 to 25 °C.
Special Requirements
Amplification Staging Room
Precautions are required when manipulating genomic DNA or setting up amplification reactions to avoid
contamination with foreign DNA amplified in other reactions and procedures.
It is highly recommended that genomic DNA manipulations and amplification reaction set up are
performed in a dedicated amplification staging room separate from the main laboratory. This
amplification staging room should have a dedicated set of pipettes and plasticware.
If no dedicated amplification staging room is available, use of a dedicated bench or a dedicated biosafety
hood and dedicated pipettes is suggested.
If no dedicated bench is available, a set of dedicated pipettes is recommended.
Fume Hood
At certain steps in the protocol we recommend the use of adequate local or general ventilation to keep
airborne concentrations low.
A fume hood is suggested as a way to achieve the desired concentration. Thus a fume hood is strongly
recommended for several steps of this assay.
Safety Warnings and Precautions
CAUTION: All chemicals should be considered as potentially hazardous. We, therefore,

recommend that this product is handled only by those persons who have been trained in
laboratory techniques and that it is used in accordance with the principles of good laboratory
practice. Wear suitable protective clothing, such as lab coat, safety glasses and gloves. Care
should be taken to avoid contact with skin and eyes.
WARNING: The following components contain harmful or toxic ingredients:

Axiom Stabilize Soln: 8% Gluteraldehyde
Axiom HybSoln 2: 100% Formamide

Axiom Hyb Buffer: <55% Tetramethylammonium Chloride
As such we recommend the use of a fume hood when using these products during the Manual
Target Preparation protocol. In all cases customers should use adequate local and general
ventilation in order to minimize airborne concentrations.
Copies of the Material Safety Data Sheets for the kit components are available on the Affymetrix website
at www.affymetrix.com
Control Recommendations
A negative control is not required for this assay.
We recommend including one positive control with every set of samples processed. A positive control
(Axiom Reference Genomic DNA 103) is included in the Affymetrix® Axiom Reagent Kit.
Plate Requirements and Recommendations

The following types of plates are required for performing manual target preparation:
ABgene 96 Square Well Storage Plate, 2.2 mL
Chapter 4 | Axiom Genotyping Assay: Manual Target Preparation 97
Bio-Rad Hard Shell 96-well plate, p/n HSP9631\Refer to the Axiom Site Prep Guide, P/N 702858, for
vendor information.
96-well UV Star Plates, 370 µL/well
Thermal Cycler Recommendations

The following thermal cyclers are recommended:
BIO-RAD PTC200 or
Whatman Biometra TRobot
BIO-RAD MJ 0240G
ABI 9700
ABI 2720
NOTE: The ABI 9700 and the ABI 2720 use the half-skirted 96-well plates (PN HSS-9601)
IMPORTANT: Always use the heated lid option when programming protocols.
We have verified the performance of this assay using the following thermal cyclers: Bio-Rad PTC-200,
Biometra TRobot 96, ABI 9700 with a gold, silver or aluminum block (ABI 2720 and the Bio-Rad MJ
0240G). The performance of this assay has not been verified with other thermal cyclers. Use of other
thermal cyclers may result in assay failure and may violate the Axiom Array and Reagent replacement
policy. The thermocycler needs to be programmed with the “Axiom Denature” protocol:
1. 95 °C 20 min
2. 48 °C 3 min
3. 48 °C hold
Use the heated lid option when setting up or running the protocol.
WARNING: Evaporation during denaturation can negatively impact assay performance. Use
the recommended thermal cycler consumables and sealing film to eliminate condensation
and evaporation. The arched, auto-sealing metal plate with P pads (used with the TRobot
thermal cycler) as shown in Table 3.2 on page 22 should be replaced after use as per the
manufacturers recommendation.
Thermal cycler Consumables

Table 4.1 provides details into the consumables to be used with each thermal cycler.
Table 4.1 Thermal Cycler Consumables for Axiom

Thermal Cycler Model PCR Plate Type Seal*
Bio-Rad PTC-200 Bio-Rad Hard-Shell Thin-Wall 96-Well MicroAmp Clear Adhesive Film from
Skirted PCR Plates, P/N HSP9631 Applied Biosystems (p/n 4306311)
TRobot Bio-Rad Hard-Shell Thin-Wall 96-Well BioRad Arched Auto-Sealing Lids

Skirted PCR Plates, P/N HSP9631 withWide Tabs (p/n MSL-2032) with
BioRad Micro seal 'P' Replacement Pads
(MSP-1003)
ABI 9700 Bio-Rad P/N HSS-9601 (half skirted plate) MicroAmp Clear Adhesive Film from
Applied Biosystems (p/n 4306311)
ABI 2720 Bio-Rad P/N HSS-9601 (half skirted plate) MicroAmp Clear Adhesive Film from
Applied Biosystems (p/n 4306311)
Bio-Rad Tetrad 2 PTC-0240 Bio-Rad Hard-Shell Thin-Wall 96-Well MicroAmp Clear Adhesive Film from
Skirted PCR Plates, P/N HSP9631 Applied Biosystems (p/n 4306311)
* Microseal "B" film from BioRad (p/n MSB-1001) may be used in place of MicroAmp Clear Adhesive Film for the BioRad and ABI thermal cyclers.
Oven Recommendations
The following ovens are recommended:
ED 53 drying oven by Binder (supplier VWR)
Specifications:
for the 115 V version:

50/60 Hz, 115 V, 1200W
VWR P/N 47746-744
Mfg P/N 9010-0131
for the 230 V version:
50/60 Hz, 230 V, 1200 W
VWR P/N 47746-690
Mfg P/N 9010-0078
Affymetrix GeneChip Hyb Oven 640 or 645
If using either Affymetrix GeneChip Hyb Oven, set the rotation speed to 15 RPM to aid in even heat
distribution.
For either Affymetrix GeneChip Hyb Oven, plates are placed in the bottom of the oven. To avoid
interfering with the rotation apparatus, do not stack plates in the oven.
Up to 2 plates can fit in a Hyb Oven 640
Up to 4 plates can fit into a Hyb Oven 645
For the Hyb Oven 640, place the plate away from the heat vent.
Multiple ovens are required for manual target preparation. The exact number depends upon whether you
are running only a single sample plate and array plate through the workflow, or if you are trying to run
the three plate/week manual target preparation workflow.
If you are running individual plates, you will need two ovens for the workflow.
If you are running the three plate /week workflow, you will need three ovens.
See Changing Oven Temperatures for the Three Plate Workflow on page 211 of Chapter 8, Manual
Target Preparation for Processing Three Axiom Array Plates per Week for more information.
Equipment Care and Calibration

Lab instrumentation plays an important role in the successful completion of this assay. To aid in
maintaining consistency across samples and operators, all equipment must be regularly calibrated and
well maintained, including:
All pipettes, thermal cyclers, and ovens
Plate spectrophotometer
Procedures
This section covers procedures you may need to do repeatedly during the workflow, or which are critical
to the performance of the assay.
Seal, Vortex and Spin

Unless otherwise noted, when the protocol instructs you to seal, vortex and spin:
Seal plates — we recommend using MicroAmp Clear Adhesive Films to seal your plates.
IMPORTANT: Always ensure that your plates are tightly sealed. A tight seal will prevent
sample loss and cross-well contamination, particularly when plates are being vortexed.
Spin — when instructed to perform a brief spin down of plates or reagent vials, follow these guidelines
unless otherwise instructed.
Plates:
• Spin at room temperature.

• Start the centrifuge, allow it to reach 1000 rpm and spin for 1 min.
Reagent Vials: 3 sec
Vortex reagents 3 times, 1 sec each time.

Vortex plates 1 to 2 sec each sector for a total of 5 sectors (Figure 4.1).
Figure 4.1 Vortexing plates
1 3
2 4
NOTE: In the procedures, “vortex twice” means to repeat the vortexing step.
Sample Quantitation
This protocol has been optimized using a PicoGreen assay to determine genomic DNA concentrations.
Other quantitation methods such as UV Absorbance may give different readings. Therefore, you should
correlate readings from other methods to the equivalent PicoGreen-determined concentration.
Please refer to Chapter 2, Genomic DNA Preparation and Requirements on page 9 for more information.
About the Reagents and Master Mix Preparation

Axiom Reagent Kit Components
Caps on the vials are color-coded by assay stage.
Properly store all enzyme reagents, especially enzyme-containing vials. Improper storage methods can
profoundly impact activity.
IMPORTANT: Use only the reagents from the Axiom Reagent Kit for this assay. These
reagents are not interchangeable with reagents from other Affymetrix reagent kits.
Reagents from Other Suppliers

Use only fresh reagents from the recommended vendors to help eliminate changes in pH or the salt
concentration of buffers.
Consult the appropriate MSDS for reagent storage and handling requirements.
Master Mix Preparation
Carefully follow each master mix recipe. Use pipettes that have been calibrated to ± 5%.
If you run out of master mix during any of these procedures, a volume error has been made or the
pipettes are not accurate. We recommend that you stop and repeat the experiment.
NOTE: The volumes of Master Mixes prepared are designed to provide consistent handling of
reagents and consistent assay results. The percent overage of different master mixes may
differ, depending upon the reagent volumes involved.
When Using Reagents at the Lab Bench

Properly chill essential equipment such as reagent coolers before use.
Ensure that enzymes are kept at –20 °C until needed. When removed from the freezer, immediately
place in a cooler that has been chilled to –20 °C.
Pipettes and Pipetting

To efficiently process samples:
Use 12-channel pipettes to transfer samples between plates and to add Master Mix to plates or
GeneTitan Trays
Use single-channel pipettes for preparing Master Mixes and for puncturing bubbles in GeneTitan
Trays. The single-channel pipettes will not be used for working with the plates or trays otherwise.
Use a pipette of appropriate size for the volume of liquid being transferred (Table 4.2).
Table 4.2 Recommended pipette sizes

Pipette Size Recommended volume Range
P20 1-20 uL
P50 (optional) 20-50 uL
P200 20-200 uL
Single channel P1000 / 200-1000 uL

12-channel P1200
We recommend the use of Rainin pipettes and tips. Affymetrix has only verified the use of Rainin multi
channel pipettes in this assay. The use of other pipettes (such as other brands or 8-channel pipettes)
may impact the timing of the protocol and may adversely impact the assay. Pipette substitution may
violate the terms of the Axiom Assay and Array Replacement policy.
Always use pipettes that have been calibrated to ± 5%.
It is essential that you be proficient with the use of single- and multi-channel pipettes.
To familiarize yourself with the use of multi-channel pipettes, we strongly recommend practicing
several times before processing actual samples. Use water and solution basins to get a feel for
aspirating and dispensing solutions to multiple wells simultaneously.
Single-channel Pipettes and Serological Pipettes
Use single-channel pipettes for preparing Master Mixes and for puncturing bubbles in GeneTitan Trays.
The single-channel pipettes will not be used for working with the plates or trays otherwise.
Use single channel pipettes for volumes less than or equal to 2 mL. For volumes between 1 and 2 mL,
add the reagent in two portions with a fresh tip for each portion.
Use serological pipette for volumes > 2 mL.
In most cases, 25 or 50 mL serological pipettes will not fit into the mouths of the reagents bottles.
Multiple transfers using 5 or 10 mL serological pipettes will need to be performed.
Multi-Channel Pipettes
Use 12-channel pipettes when working to add Master Mix or to transfer samples to plates and GeneTitan
trays.
Use a pipette of appropriate size for the volume of liquid being transferred.
Change pipette tips after each transfer or addition
GeneTitan MC Consumables
All consumables for the GeneTitan MC Instrument are provided by Affymetrix. The following table
provides guidance on the consumables that are shipped with the Array Plate.
IMPORTANT: All GeneTitan trays and tray covers must have barcodes. Discard any
consumable tray or tray cover without a barcode.
Table 4.3 Axiom GeneTitan Tray consumables

HT Array All array plates The HT Array Plate

Plate have the PN shipping package
Package 202091 etched includes the following:
on the on the The function of the clear
plastic plastic cover for the
Array Plate is to protect
the Array Plate during
transport. You can
discard this after
removing the Array
Plate.
The Array Plate must be
from damage or
Array Plate must be in
the Blue Array Plate
Protective Base at all
times.
The Blue Array Plate
Protective Base in the
package must be used
to protect the Array
Plate from damage.

The HT Scan Tray

shipping package
includes the following:
The HT Scan tray
includes a Scan Tray
cover. The tray cover
should be used to cover
the scan tray before
placing the tray in the
GeneTitan MC
Instrument. The Tray
cover should be de-
ionized before use. See
the section Appendix E,
the anti-static
procedure.
The Scan tray must be
from damage or
Scan Tray must be in the
Black Plate Cover at all
times.
The Black Scan tray
cover in the package is
used to protect the scan
tray glass from damage.
used with the Array
Plate. Remove the
the scan tray with the
scan tray cover
Black Scan PN 202096 The Black Scan tray

Tray cover in the package is
protective used to protect the scan
base tray glass from damage.
used with the Array
Plate.
Remove and discard the

Scan Tray Scan Tray 501006 The GeneTitan Scan tray

with cover Cover 202757 must be loaded with the
scan tray cover into the
Do not load the scan
tray with the protective
base
GeneTitan 501025 The GeneTitan stain trays

Stain Trays are packaged in ziplock
bags to keep them free
of dust. The GeneTitan
stain trays are barcoded
and tray have separator
walls that are flush with
the frame of the stain
tray as shown by the
yellow line.

GeneTitan 202757 The GeneTitan scan and

Scan and stain tray covers are
Stain Tray provided to prevent any
cover evaporation of the
stains in stain trays and
the array holding buffer
in the scan tray
All stain and scan trays
must be placed in the
with the GeneTitan stain
tray cover.
All tray covers must be
de-ionized to remove
static electricity prior to
placing the cover on the
tray.
See the section
the anti-static
procedure.
GeneTitan Tray 501025 The GeneTitan stain

stain tray Cover 202757 trays must be placed in
shown with the GeneTitan
the stain instrument with the
tray cover GeneTitan stain tray
cover.
It is important to remove
the static electricity on
the cover and the stain
tray prior to loading the
tray into the GeneTitan
instrument.
See the section
the anti-static
procedure.
GeneTitan 500867 The GeneTitan

Hyb Tray hybridization trays are
packaged in white
pouches with the label
“HT Hybridization
Tray.”
After aliquoting the hyb
cocktail into the
hybridization tray, the
tray should be loaded
into the GeneTitan
instrument with the
barcode facing away
from the operator, i.e.,
Barcode should be on
the back side.

Figure 4.2 Labeling GeneTitan Hyb Trays
Writing on the wrong side of the Hyb tray, or on the wrong part of the long side, may interfere with the
operation of sensors in the GeneTitan MC.
You may label the Stain trays on the left side of the front of the tray as shown in Figure 4.3. The correct
Figure 4.3 Labeling GeneTitan Stain Tray
(see Stage 5 — Manually Preparing Ligation, Staining, and Stabilization Reagent Trays for the
GeneTitan MC Instrument on page 134 for detailed information).
Stage 1 — DNA Amplification
IMPORTANT: Before proceeding to DNA Amplification, perform the gDNA preparation

described in Chapter 2, Genomic DNA Preparation and Requirements on page 9.
NOTE: For this protocol, the term samples includes the positive control.
The following sets of steps are necessary to perform DNA amplification:

1: Initial Setup for DNA Amplification on page 109
2. Prepare the Denaturation Master Mix and the Neutralization Master Mix on page 111
3. Add Denaturation Master Mix to Samples on page 112
4. Add Neutralization Master Mix to Samples on page 113
5. Prepare and Add the Amplification Master Mix on page 113
6. Freeze or Proceed on page 115
IMPORTANT: Amplification preparation should take place in an amplification staging room

or dedicated area such as a biosafety hood with dedicated pipettes, tips, vortex, etc. See
Amplification Staging Room on page 96 for more information.
Duration
For 96 samples:
Time to thaw materials: 1 hr

Hands-on time: approximately 0.5 hr
Incubation at 30 °C: 23 ± 1 hr
Total time required: approximately 24.5 hr
Input Required
gDNA Sample Plate, with 20 µL of each gDNA diluted to a concentration of 10 ng/µL.
See Genomic DNA Preparation on page 11 for more information.

Table 4.4 Equipment and consumables required for Stage 1 — DNA Amplification.
Quantity Item
As required Adhesive seals for 96-well plate
1 Metal chamber for 50 mL tubes, chilled to 4 °C on ice (do not

freeze)
1 Cooler, chilled to –20 °C
1 15 mL tube holder

Table 4.4 Equipment and consumables required for Stage 1 — DNA Amplification.
Quantity Item
1 each Pipettes:
Single-channel P100
Single-channel P200
Single-channel P1000
Multi-channel P20
Multi-channel P50 or P200
Multi-channel P1200
As needed Pipette tips

(Use tips manufactured specifically for your brand of pipette.)
As needed Pipette, serological

5 x 1/10 mL (VWR P/N 53283-706)
10 x 1/10 mL (VWR P/N 53283-708)
1 Pipet-aid
1 Plate centrifuge, at room temperature
1 Oven, set at 30 °C
1 50 mL Falcon tube
2 15 mL Falcon tube
1 Vortexer
1 Timer
As needed Solution basin, 100 mL sterile multichannel
Reagents Required
Table 4.5 Reagents required for Stage 1 — DNA Amplification on page 107
Axiom Reagent Kit Module

Module 1, –20 °C
Axiom Amp Soln
P/N 901275
Axiom Amp Enzyme
Axiom Water
1: Initial Setup for DNA Amplification

In this set of steps you will:
Set temperatures for the oven and centrifuge
Label the Master Mix tubes and solution basins
Thaw the reagents and sample plate.
To perform the initial setup
1. Set an incubator/oven temperature at 30 °C.
2. Set the centrifuge temp to room temperature.
3. Prepare reagents as shown in Table 4.6:
NOTE: Leave the Axiom Amp Enzyme at -20 °C until ready to use.
Table 4.6 Initial Preparation of reagents for Amplification

Reagent Temp Out of Module* Treatment Store before using in
Master Mix
Axiom Amp Soln Thaw at Room Temp Vortex twice Place on ice
(~1 hr)
(see note below)
Axiom Water Thaw at Room Temp Vortex Keep at Room Temp
Axiom Denat Soln Thaw at Room Temp Vortex and spin Keep at Room Temp
Axiom Neutral Soln Thaw at Room Temp Vortex and spin Keep at Room Temp
Axiom Amp Enzyme Soln Keep at -20 °C Just before use, flick tube 3X, Keep in -20 °C cooler until
spin, and place in -20 °C ready to use
portable cooler
Notes:
*Temp Out of Module: temperature reagent is held at immediately after removal from module
NOTE: Allow ~ 1 hour for Axiom Amp Soln to thaw on the benchtop at room temperature.
If the solution is not completely thawed after 1 hour, vortex briefly and return to the
benchtop to complete thawing.
IMPORTANT: The Axiom Amp Soln must be thoroughly mixed before use. Place on ice
immediately after thawing.
4. Thaw Samples in gDNA Plate:

A. Bring your gDNA samples to room temperature on the bench top.
B. Vortex and spin.
C. Leave at room temperature.
IMPORTANT:
gDNA samples must be brought to room temperature before proceeding with
denaturation.
gDNA samples must be 20 μL volume of each gDNA at a concentration of 10 ng/μL (see
Genomic DNA Preparation on page 11).
5. Label the 15 mL and 50 mL Falcon tubes as indicated in the table below:

i
Label Tube Size Temperature Contents
D MM 15 mL leave tube at room temperature Axiom Denaturation Master Mix
N MM 15 mL leave tube at room temperature Axiom Neutralization Master Mix
Amp MM 50 mL place tube on ice Amplification Master Mix

NOTE: It is recommended to use a chilled metal chamber for 50 mL tubes to stabilize the Amp
MM tube in the ice bucket.
6. Label three solution basins as indicated in the table below.

i
Label Temperature Contents
D MM Leave basin at room temperature Axiom Denaturation Master Mix
N MM Leave basin at room temperature Axiom Neutralization Master Mix
Amp MM Place basin on ice Amplification Master Mix
A suggested bench setup for denaturation is shown in the figure below.
Figure 4.4 Work area prepared for denaturation
Axiom Denat Soln

and Neutral Soln
15 mL tubes for Vortexer

D MM and N MM
Timer
Axiom
Water
D MM Tips
Solution
Basins
gDNA
N MM Sample Plate
2. Prepare the Denaturation Master Mix and the Neutralization Master Mix
Preparing the Denaturation Master Mix

To prepare the Denaturation Master Mix:
1. Per Table 4.7 on page 112, dilute the appropriate volume of Axiom Denat Soln 10X using the Axiom
Water.
Table 4.7 Preparing Axiom Denaturation Master Mix (D MM)

Reagent per sample Master Mix 96+
To the 15 mL tube marked D MM, add:
Axiom Denat Soln 10X 2 μL 300 μL
Axiom Water 18 μL 2.7 mL
Total Volume 20μL 3 mL
2. Vortex and leave at room temperature.
Preparing the Neutralization Master Mix

To prepare the Neutralization Master Mix:
1. Per Table 4.8, dilute the appropriate volume of Neutral Soln 10X using the Axiom Water.
Table 4.8 Preparing Axiom Neutralization Master Mix (N MM)

To the 15 mL tube marked N MM, add:
Axiom Neutral Soln 10X 4 μL 500 μL
Axiom Water 36 μL 4.5 mL
Total Volume 40 μL 5 mL
2. Vortex and leave at room temperature.
3. Add Denaturation Master Mix to Samples

To add the Denaturation Master Mix to your samples:
1. Spin down the Sample plate.
Remember: Samples must be at room temperature for this step.
2. Pour the Denaturation Master Mix into the solution basin marked D MM. Leave the solution basin on
the benchtop.
3. Carefully remove the seal from the Sample plate and discard the seal.
4. Using a P20 12 channel pipette and pipetting directly into the liquid of each well, add 20 µL of
Denaturation Master Mix to each sample of the plate (total volume 40 µL/well).
Change tips between each addition.
NOTE: Pipette directly into the liquid of the well; do not mix by pipetting up and down.
NOTE: This plate is now known as the Denaturation plate.
5. Seal and vortex the Denaturation plate. Start the timer for 3 minute incubation.
6. Do a quick spin on the Denaturation plate in a room temperature centrifuge by bringing centrifuge
speed to 1000 rpm (takes ~ 1 minute).
NOTE: The quick spin time is included in the 3 minute incubation.
7. Visually examine the volume in each well (should be 40 µL/well) and:

A. Keep a record of any wells that visually appear to have a particularly low or high volume; these
samples may need to be repeated.
B. Do NOT stop to measure volumes; proceed without delay.
8. Complete the 3 minute incubation on the benchtop at room temperature.
While completing the incubation at room temperature, pour the Neutralization Master Mix into the
solution basin as described in Step 1 on page 113.
9. After incubation immediately add the Neutralization Master Mix as described in 4. Add
Neutralization Master Mix to Samples on page 113.
4. Add Neutralization Master Mix to Samples

To add the Neutralization Master Mix to your samples:
1. Pour the Neutralization Master Mix into the solution basin marked N MM. Leave the solution basin
on the benchtop.
2. Carefully remove the seal from the Denaturation plate and discard the seal.
3. Using a P50 or P200 12 channel pipette, pipetting directly into the liquid of each well, add 40 µL of
Neutralization Master Mix to each sample (total volume 80 µL/well).
NOTE: Pipette directly into the liquid of the well; do not mix by pipetting up and down.
NOTE: This plate is now known as the Neutralization plate.
4. Seal, vortex, and spin the Neutralization plate.

5. Visually examine the volume in each well (should be ~80 µL/well) and:
A. Keep a record of any wells that visually appear to have a particularly low or high volume; these
samples may need to be repeated.
B. Do NOT stop to measure volumes.
6. Place the Neutralization plate on ice.
7. Proceed immediately to 5. Prepare and Add the Amplification Master Mix on page 113.
5. Prepare and Add the Amplification Master Mix

A suggested ice bucket setup for Amplification is shown in the figure below.
Figure 4.5 Ice bucket set up for Amplification
Metal Chamber
for 50 mL tubes Axiom Amp Enzyme
in –20 °C cooler
Solution
Basin
Amp MM
tube
Neutralization
Plate
Axiom Amp Soln and

Axiom Water
IMPORTANT: Complete this step as efficiently as possible and with minimal delays. Once the
Amp Enzyme is added to the master mix, finish this step with no delays.
To prepare and add the Amplification Master Mix:

1. Place the 50 mL tube labeled Amp MM on ice.
NOTE: It is recommended to use a chilled metal chamber for 50 mL tubes to stabilize the Amp
MM tube in the ice bucket.
2. Per Table 4.9, pipette the appropriate amount of Axiom Amp Soln into the 50 mL tube labeled Amp
MM in the chilled metal chamber.
NOTE: Use a 10 mL serological pipette to transfer Axiom Amp Soln to the tube. The bottles
have narrow openings, and a 25 mL pipette will not fit through the mouth of the bottle.
Table 4.9 Amplification Master Mix (Amp MM)

Reagent Per Sample (μL) Master Mix 96+
To the 50 mL tube marked Amp MM, add:
Axiom Amp Soln 304 μL 34 mL
Axiom Amp Enzyme 16 μL 1.79 mL
Total Volume 320 μL 35.79 mL
3. Remove the Axiom Amp Enzyme from the freezer and place in a portable cooler at –20 °C.
A. Flick the Axiom Amp Enzyme tube three times, then spin.
B. Per Table 4.9 on page 114, add the appropriate amount of Axiom Amp Enzyme to the tube labeled
Amp MM and place on ice.
4. Moving through the remaining steps without delay:

A. Vortex the Amplification master mix well, then invert the tube 2 times, and then vortex again.
B. Quick spin and return the Amp MM tube to the ice bucket.
C. Place the solution basin labeled Amp MM on ice.
D. Slowly pour the Amplification Master Mix to the solution basin labeled Amp MM.
IMPORTANT: Keep the Amplification master mix solution basin and Neutralization
plate on ice throughout this procedure.
E. Carefully remove the seal from the Neutralization plate and discard the seal.
F. Using a P1200 12 channel pipette, slowly add 320 µL Amplification Master Mix to each well of
the Neutralization plate, pipetting down the wall of the well (there will now be a total volume of
400 µL/well).
Do not mix by pipetting up and down.
NOTE: After adding the Amplification Master Mix, the plate is now known as the
Amplification plate.
G. Seal, vortex twice, and spin the Amplification plate for one minute at 1000 rpm.
H. Place the sealed amplification plate in an oven set at 30 °C and leave undisturbed for 23 ± 1 hr.
oven. Plates do not rotate. Set the rotor for 15 rpm speed. See Oven Recommendations
on page 98 for more information.
After the incubation finishes, you can either:
Proceed to Stage 2 — Fragmentation and Precipitation on page 115.
Store the amplification plate at –20 °C.
NOTE: If freezing, do not perform the stop amplification reaction step before you store the
Amplification plate at –20 °C. The Stop Amplification Reaction step will be performed after
thawing the frozen plate, as described in 1: Stop Amplification Reaction on page 117.
Stage 2 — Fragmentation and Precipitation

The following sets of steps are necessary to perform fragmentation and precipitation:
1: Stop Amplification Reaction on page 117
2: Prepare Fragmentation Master Mix on page 119
3: Add Fragmentation Master Mix to Wells on page 119
4: Aliquot the Stop Solution to the Fragmentation Plate on page 120
5: Prepare and Add Precipitation Master Mix on page 121
Duration
Total time: approximately 2 hours.
Input Required
Amplification plate from Stage 1 — DNA Amplification on page 107.

Table 4.10 Equipment and consumables required for Stage 2 — Fragmentation and Precipitation
Quantity Item
As required Adhesive seals for 96-well plates
1 Metal chamber for 50 mL tubes, chilled to 4 °C (do not freeze)
1 Freezer set to –20 °C (Designate a shelf where the precipitation plates

can be left undisturbed)
1 Cooler, chilled to –20 °C
1 each Pipettes:
Single channel P1000
Single channel P200
Multi-channel P200
Multi-channel P1200
As needed Pipette tips for pipettes listed above
(Use tips manufactured specifically for your brand of pipette.)

Table 4.10 Equipment and consumables required for Stage 2 — Fragmentation and Precipitation
Quantity Item
1 Pipet-aid
1 Plate centrifuge set at room temp
2-3 Ovens (see Oven Recommendations on page 98):

One oven set at 37 °C
One oven set to 65 °C
1 15 mL Falcon Tube
1 50 mL Falcon Tube
4 Solution basin, 100 mL sterile multichannel
1 Vortexer
Reagents Required
Table 4.11 Reagents required for Stage 2 — Fragmentation and Precipitation

Reagent Module
From the Axiom Reagent Kit 96 reactions: 901281
Axiom Frag Enzyme (leave at –20 °C until ready to use)

Module 2
Axiom 10X Frag Buffer Box 1, –20 °C
P/N 901528
Axiom Precip Soln 2
Axiom Frag Diluent

Module 2
Axiom Frag Rxn Stop Box 2, 2–8 °C
P/N 901529
Axiom Precip Soln 1
User-supplied
Isopropanol, 99.5% 96 samples: 65 mL
1: Stop Amplification Reaction

If you are running one plate per week, you will need two ovens to perform this step:
One oven which will have the temperature changed between 30 °C and 37 °C, depending upon the step
being performed.
Use an oven that can sustain a constant temperature of 37 °C and has a temperature accuracy of
+/– 1 °C.
One oven set at 65 °C.
If you are running the three plate per week manual target preparation workflow, you will need three
ovens. See Chapter 8, Manual Target Preparation for Processing Three Axiom Array Plates per Week on
page 209 for more information.
NOTE: If the plate has been frozen and stored, it must be thawed using the instructions in
Thawing Frozen Plates of Amplified DNA on page 212. Allow an hour to thaw.
To stop the amplification reaction:

1. Place the Amplification plate in the 65 °C oven:
If proceeding directly from the end of Stage 1 — DNA Amplification on page 115, transfer the
Amplification plate from the 30 °C oven to the 65 °C oven and incubate for 20 minutes.
If working with a thawed plate, place the thawed Amplification plate in the 65 °C oven and incubate
for 20 minutes.
2. If using two ovens, set the 30 °C oven previously used for amplification to 37 °C.
3. Prepare reagents as shown in Table 4.12 at the start of the 65 °C incubation of the amplification plate.
NOTE: Leave the Axiom Frag Enzyme at –20 °C until ready to use.
Table 4.12 Reagent Preparation for Fragmentation and Precipitation

Reagent Temp Out of Treatment Store before using in
Module* Master Mix
Axiom 10X Frag Buffer Thaw at Room Temp Vortex Place on ice
Axiom Frag Diluent Place on ice Vortex and spin Place on ice
Axiom Frag Enzyme Keep at -20 °C Just before use, flick tube 3X, Keep in -20 °C cooler until
spin, and place in -20 °C ready to use.
portable cooler
Axiom Frag Rxn Stop Room Temp Vortex Keep at Room Temp
Precip Soln 1 Place on ice Vortex Place on ice
Precip Soln 2 Thaw at Room Temp Vortex and spin Place on ice
Isopropanol Room Temp Room Temp Room Temp
Notes:
4. Optional: Remove samples for quantifying amplification yield by the PicoGreen Assay.
A. Carefully remove the seal from the Amplification plate and discard the seal.
B. Transfer 4 µL samples from each well to a Bio-Rad Hard Shell 96-well plate, HSP-9631.
C. Quantitate each sample (e.g., using the Quant-iT ™ PicoGreen® dsDNA Kit).
D. Reseal the Amplification plate.
5. Transfer the Amplification plate from the 65 °C oven to the 37 °C oven and incubate for 45 minutes.
6. Set the plate centrifuge to room temperature.
TIP: Keep a labeled balance plate of equal weight ready to minimize any time delay
before spinning the Fragmentation plate during later steps.
7. Label the 15 mL and 50 mL Falcon tubes as indicated in the table below:

i
Frg MM 15 mL Place tube on ice Axiom Fragmentation Master Mix
Precip MM 50 mL Place tube on ice Axiom Precipitation Master Mix

8. Label solution basins as indicated in the table below.

i
Frg MM Leave basin at room temperature Axiom Fragmentation Master Mix
Stop Leave basin at room temperature Axiom Frag Rxn Stop
Precip MM Place basin on ice Axiom Precipitation Master Mix
ISO Place basin on ice Isopropanol
2: Prepare Fragmentation Master Mix

To prepare the Fragmentation Master Mix:
1. Start making the fragmentation master mix when there is still five minutes to the finish of the 37 °C
incubation, using the values in the table below.
Transfer the Axiom Frag Enzyme to a -20 °C portable cooler until ready to use.
Table 4.13 Axiom Fragmentation Master Mix

To the 15 ml tube marked Frg MM, add:
Axiom 10X Frag Buffer 45.7 µL 5.3 mL
Axiom Frag Diluent 10.05 µL 1.17 mL
Axiom Frag Enzyme 1.25 µL 145.0 µL
Total Volume 57 µL 6.62 mL
Add the reagents from Table 4.13 to the Frg MM tube in the order shown, using appropriate single
channel and serological pipettes.
Just before the end of the 45 minute 37 °C incubation, flick the Axiom Frag Enzyme tube 2 to 3 times,
and spin
Add the Axiom Frag Enzyme to the Fragmentation Master Mix at the end of the 45 minute 37 °C
incubation.
NOTE: Leave the Axiom Frag Enzyme at –20 °C until ready to use.
2. Vortex twice and place on ice.

3. Slowly pour the Fragmentation Master Mix in the solution basin labelled Frg MM placed at room
temperature.
3: Add Fragmentation Master Mix to Wells
IMPORTANT: Work quickly to perform this set of steps to minimize the time that the
Fragmentation plate is out of the 37 °C oven.
1. Carefully remove the Amplification plate from the 37 °C oven and place on the bench top at room
temperature.
Do not place the Amplification plate on ice.
2. Carefully remove the seal from the Amplification plate and discard the seal.
3. Pipetting directly into the liquid of each well, use a P200 12 channel pipette to add 57 µL of
Fragmentation Master Mix to each reaction.
Change tips after each addition.
NOTE: After adding the Fragmentation Master Mix to the plate, the plate is now known as
the Fragmentation plate.
4. Seal the Fragmentation plate and vortex twice.

5. Start the timer for 30 min.
6. Spin the Fragmentation plate in the plate centrifuge at room temperature by bringing the centrifuge
to 1000 rpm and stopping it.
IMPORTANT: Keep your timer in a safe place. It is helpful to note down the actual time
when the incubation began in case the timer stops accidentally.
7. Quickly transfer plate to 37 °C oven and incubate for 30 min.
CAUTION: Be watchful for the end of the thirty minute incubation period.
Fragmentation is an exact 30 minute incubation step. Longer and shorter incubation
times may lead to poor performance of the assay.
Prepare the Stop solution a few minutes before the end of the 30 minute incubation period, as
described in 4: Aliquot the Stop Solution to the Fragmentation Plate, below.
4: Aliquot the Stop Solution to the Fragmentation Plate

To add the stop solution:
1. A few minutes before the end of the 30 minute incubation period, pour the Axiom Frag Rxn Stop
solution in the solution basin labelled Stop.
Leave the Stop solution basin at room temperature.
2. Remove the Fragmentation plate from the oven and place on the bench top.
3. At the end of the 30 minute fragmentation incubation period, carefully remove the seal from the
Fragmentation plate and discard the seal.
4. Using a P20 12-channel pipette, end the fragmentation reaction by adding 19 µL of Stop Solution to
each reaction.
Pipette directly into the liquid of each well.
Proceed immediately to the next step.
5. Seal and vortex twice and do a quick spin at 1000 rpm.

6. Place the Fragmentation plate on ice while you prepare the Precipitation Master Mix.
5: Prepare and Add Precipitation Master Mix

To prepare and add Precipitation Master Mix:
1. Prepare Precipitation Master Mix in the 50 mL Falcon tube labelled Precip MM
A. Add the reagents in the order and volumes shown in Table 4.14, using the appropriate serological
pipette and single-channel pipette. The tip of a 25 or 50 mL serological pipette will not fit through
the narrow opening of the bottles.
Table 4.14 Axiom Precipitation Master Mix

To the 50 mL tube marked Precip MM, add:
Axiom Precip Soln 1 238 μL 26 mL
Axiom Precip Soln 2 2 μL 218 μL
NOTE: Use a 5 or 10 mL serological pipette to pipette Axiom Precip Soln 1. The bottle
has a narrow opening and a 25 mL serological pipette will not fit through the mouth
of the bottle.
B. Vortex the Precip MM tube and place on ice.
TIP: It is recommended to place the Precip MM tube in the chilled 50 mL metal chamber
and put the chamber on ice.
2. Place the Precip MM solution basin on ice.

3. Pour the Precipitation Master Mix into the solution basin labeled Precip MM.
4. Carefully remove the seal from the Fragmentation plate and discard the seal.
5. Using a P1200 12-channel pipette, add 240 µL Precipitation Master Mix to each sample. Rest each
pipette tip against the wall of each well while delivering. You do not need to mix up and down.
NOTE: After adding the Precipitation Master Mix, the plate is now known as the
Precipitation plate.
6. Seal the Precipitation plate and vortex twice. Spin.

7. Remove the Precipitation plate from the centrifuge and place on ice.
8. Place the solution basin labeled ISO on ice and pour the isopropanol into it.
9. Carefully remove the seal from the Precipitation plate and discard the seal.
10. Using a P1200 12-channel pipette, add 600 µL isopropanol to each sample and mix well by pipetting
up and down within the solution to ensure mixing. The solution should look homogenous in the tips
after pipetting 5-7 times.
If not, repeat mixing a few more times until the solution looks mixed.
Change the tips after each addition.
11. Blot the top of the plate with Kimwipe and seal tightly with a Microamp seal.
12. Carefully transfer the Precipitation plate into the -20°C freezer and incubate overnight (16-24 hours).
.
TIP: It is recommended to designate a shelf in a -20 C freezer where the plates can be left
undisturbed.
Stage 3 — Drying, Resuspension and QC

This stage requires the following sets of steps:
1: Centrifuge and Dry Pellets and Thaw Reagents on page 124
2: Prepare the Tubes, Basins, and Trays on page 125
3: Resuspension and Hybridization Master Mix Preparation on page 126
4: Recommended: Perform Quantitation and Fragmentation QC Checks on page 127
5. Freeze or Proceed on page 128
CAUTION: Some of the steps in this stage should be performed under a fume hood.
Duration
Centrifuge and dry plates: 1 hour 15 minutes
Resuspension and hyb mix preparation: 25 min
Gel QC and OD: 45 min
total: 2.5 hr
Input Required
Precipitation plate from Stage 2 — Fragmentation and Precipitation on page 115.
Equipment, Consumables, and Reagents Required

Table 4.15 Equipment and consumables required for Stage 3 — Drying, Resuspension and QC
Quantity Item
As required Adhesive seals for 96-well plates
1 96-well cooling chamber, chilled to 4 °C (do not freeze)
1 each Pipettes:
Single channel P20
Single channel P-100
Multi-channel P20
Multi-channel P-200
Table 4.15 Equipment and consumables required for Stage 3 — Drying, Resuspension and QC
Quantity Item
As needed Pipette tips for pipettes listed above
(Use tips manufactured specifically for your brand of

pipette.)
2 Bio-Rad Hard Shell 96-well plate, HSP-9631 or any 96-well

PCR plate for making the dilutions:
QC Dilution Plate
Gel Samples Plate
1 Bio-Rad Hardshell 96 well plate, HSP 9631, for Bio-Rad PTC-

200, TRobot or Bio-Rad 0240G thermal cyclers or
Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted
PCR Plate (P/N HSS 9601 for ABI 9700 or ABI 2720 thermal
cyclers)
Hyb Ready Plate
1 OD plate: 96-well UV Star, 370 µL/well
1 Oven set at 37 °C
1 Fume Hood
1 Plate centrifuge set at 4 °C
1 Tube, Falcon 15 mL
1 10 mL Serological Pipette
1 Pipetaid
1 Shaker, either:
Titer Plate Shakers-4PL, 120V
Jitterbug
1 Vortexer
As needed Solution basin, 100 mL sterile multichannel
Reagents Required
Table 4.16 Reagents required for Stage 3 — Drying, Resuspension and QC

Reagent Module
Axiom Hyb Buffer Module 2

Box 1, –20 °C
Axiom Hyb Soln 1 P/N 901528
Axiom Resusp Buffer Module 2

Box 2, 2–8 °C
Axiom Hyb Soln 2 P/N 901529
Table 4.16 Reagents required for Stage 3 — Drying, Resuspension and QC

Reagent Module
Other Reagents Required for QC steps (optional)
TrackIt Gel Loading Buffer, 1000-fold dilution 8 mL

(see Appendix A, Fragmentation Quality Control Gel Protocol on
page 223 for dilution instructions.)
Gel Sample Plate
15 fold dilution of 25bp Invitrogen Ladder (P/N 10488-022)
Nuclease free water, ultrapure MB Grade (P/N 71786; for OD and Dilution 14 mL
Plate preparation)
Gels and Related Materials Required

At the end of this stage, verifying the fragmentation reaction is recommended. See Appendix A,
Fragmentation Quality Control Gel Protocol on page 223 for the required gel and related materials.
1: Centrifuge and Dry Pellets and Thaw Reagents
CAUTION: During this step, handle the Precipitation plate gently to avoid disturbing the
pellets. Do not bump or bang the plate.
To centrifuge and dry the pellets:

1. Turn the oven on and preheat to 37 °C.
If using an Affymetrix GeneChip Hyb Oven, set the rotation speed to 15 rpm to distribute heat.
If using the Affymetrix GeneChip Hyb Oven 640, keep the plate on the side away from the bottom
heat vent.
2. Begin thawing/warming the reagents used in this stage as shown in Table 4.17 on page 125.
3. Remove the Precipitation plate from the -20 °C freezer and centrifuge the plate at 3200 xg (or rcf) at
4 °C for 40 min.
.
WARNING: We strongly recommend that you use the Eppendorf 5810R at 3200 xg
(4000 rpm/) with:
Rotor A-4-81
Plate carrier DL 050 (rubber bottom)
If not using the Eppendorf 5810R with recommended rotor and plate carrier, centrifuge
the samples at 3200 xg (or rcf) at 4 °C.
Do not use the 5810R with the A-4-62 rotor and WO-15 plate carrier (hard bottom). Use of
this rotor and plate carrier may result in cracked plates, loss of sample, unbalanced
centrifugation, damage to the instrument and possible physical injury.
NOTE: If you are processing two plates at the same time, as in the three plate/week
manual prep workflow, you can centrifuge both plates at the same time.
4. During the centrifugation time prepare the resuspension and hybridization reagents as shown in
Table 4.17:
Table 4.17 Reagent Preparation for Resuspension and Hybridization

Reagent Temp Out of Module* Treatment Store before using in
Master Mix
Axiom Hyb Buffer Place on ice Vortex Place on ice
Axiom Hyb Soln 1 Thaw at Room Temp Vortex and spin Place on ice
Axiom Resusp Buffer Warm to Room Temp Vortex Keep at Room Temp
(~ 1 hr)
Axiom Hyb Soln 2 Place on ice Vortex and spin Place on ice
Notes:
5. Following centrifugation, empty the liquid from the Precipitation plate as follows:
A. Carefully remove the seal from the Precipitation plate and discard the seal.
B. Invert the plate over a waste container and allow the liquid to drain.
C. While still inverted, gently press the plate on a pile of Kimwipes on a bench and leave it for 5 min.
6. Turn the plate top side up and place in an oven for 20 min at 37 °C to dry.
7. If you are proceeding directly to 3: Resuspension and Hybridization Master Mix Preparation on
page 126, you can prepare the Hybridization Master Mix at this time (Step 6 on page 126).
8. After 20 min remove the plate from the oven and either:
Proceed directly to 3: Resuspension and Hybridization Master Mix Preparation on page 126, even
if some droplets of liquid remain. Leave the Precipitation plate at room temperature.
Seal if not proceeding immediately to the next set of steps.
Tightly seal the plate and store at –20 °C.
2: Prepare the Tubes, Basins, and Trays

To prepare the tubes, basins, and trays used in the procedure:
1. Label the 15 mL tube as indicated in the table below:
i
Hyb MM 15 mL On ice in Fume Hood Axiom Hybridization Master Mix
2. Label solution basins as indicated in the table below.

i
Resus Room Temperature Axiom Resusp Buffer
Hyb MM On Ice in Fume Hood Axiom Hybridization Master Mix
3. If performing the recommended QC checks, label solution basins as indicated in the table below:
i
NF H2O Leave basin at room temperature Nuclease Free Water
Loading Dye Leave basin at room temperature Loading dye

3: Resuspension and Hybridization Master Mix Preparation
NOTE: If a plate was stored at –20 °C after drying the pellets, it is recommended to allow the
plate to sit at room temperature for 1.5 hour before carrying out resuspension.
NOTE: Make sure the Axiom Resusp Buffer has equilibrated to room temperature before
adding to dry pellets in Step 3, below.
To resuspend the pellets:

1. Pour Axiom Resusp Buffer in the solution basin labeled Resus at room temp.
NOTE: If you are processing two plates at the same time, as in the three plate/week
manual prep workflow, you can add resuspension buffer to both plates at the same time
and then place them both in the shaker.
2. If the Precipitation plate has a seal on it, carefully remove the seal from the Precipitation plate and
discard the seal.
3. Using a P200 12 channel pipette, transfer 35 µL Axiom Resusp Buffer to each well of the
Precipitation plate with a dry pellet. Avoid touching pellets with tip.
Change pipette tips after each addition.
NOTE: After adding Resuspension buffer, the plate is known as the Resuspension plate.
4. Seal the Resuspension plate.

5. Put the sealed Resuspension plate on one of the following shakers:
Titer Plate Shakers-4PL: at speed 9 for 10 min
Jitterbug: at speed 7 for 10 min
CAUTION: It is recommended to perform the rest of the steps in this stage under a fume
hood.
6. While the Resuspension plate is shaking, prepare the Hybridization Master Mix in the Hyb MM 15
mL tube.
A. Keep the Hyb MM tube on ice.
B. Add the reagents in Table 4.18 to the Hyb MM tube in the order shown, using serological and
single-channel pipettes as needed.
Table 4.18 Axiom Hybridization Master Mix

To the 15 mL tube labeled Hyb MM, add:
Axiom Hyb Buffer 70.75 μL 7.9 mL
Axiom Hyb Soln 1 0.25 μL 27.8 μL
Axiom Hyb Soln 2 9 μL 1.0 mL
C. Vortex twice and place on ice

7. Inspect the Resuspension plate from the bottom. If the pellets are not dissolved, repeat Step 5.
8. Quickly spin at 1000 rpm.
9. Label a Bio-Rad Hard Shell 96-well plate, HSP-9631 as Hyb Ready [Sample ID] and keep covered
on ice.
When using the ABI 9700 or ABI 2720, you should label a Bio-Rad Hard-Shell Full-Height 96-
Well Semi-Skirted PCR Plate (P/N HSS 9601) as Hyb Ready [Sample ID] and keep covered on
ice.
10. Set a P200 12-channel pipette to 45 µL (this is slightly higher than the volume of sample in each well).
11. Using the P200 pipette, transfer the entire contents of each well of the Resuspension plate to the
labeled Hyb Ready plate on ice.
Change pipette tips after each transfer.
12. Pour the Hyb Master Mix to the solution basin labelled Hyb MM placed on ice.
13. Using a P200 12-channel pipette, add 80 µL of the Hyb Master Mix to each well of the Hyb Ready
plate.
14. Seal, vortex twice, and spin.
15. Keep the Hyb Ready plate on ice while preparing the dilutions for the QC steps, as described in the
next section. We recommend placing the Hyb Ready plate on a 96-well metal chamber sitting on ice
to stabilize it.
4: Recommended: Perform Quantitation and Fragmentation QC Checks

Before proceeding to Stage 4 — Denaturation and Hybridization, we recommend that you perform
quantitation and fragmentation quality control checks.
The QC checks requires:
2 each 100 ml solution basin
Label one basin as H2O
Label the second basin as Loading Dye
2 each Bio-Rad Hard Shell 96-well plate, HSP-9631 or any 96-well PCR plate for making the dilutions:
Label one plate as QC Diln
Label the second plate as Gel Sample
1 each 96-well UV Star, 370 µL/well plate, labeled OD

8ml Gel Loading dye:
1:1000 dilution of 6x Invitrogen TrackIt Cyan/Orange as described in Diluting the TrackIt Cyan/
Orange Loading Buffer on page 224.

14 mL of nuclease free water, P/N 71786
15 fold dilution of 25bp Invitrogen Ladder, P/N 10488-022
NOTE: Use appropriate pipettes to transfer samples. Change tips while transferring samples
from the Hyb Ready plate and the QC Dilution plate to avoid cross-contamination.
To perform the QC checks:

1. Pour nuclease free water into the solution basin labeled H2O. The water will be used to make the QC
Dilution plate and the OD plate.
2. Make QC Dilution Plate:
A. Add 33 µL nuclease-free water to each well of the QC Diln plate.
B. Transfer 3 µL of the Hyb Ready sample from each well of the Hyb Ready plate to the
corresponding well of the QC Diln plate.
C. Seal, vortex twice, and spin.
3. Make OD Sample plate:

A. Carefully remove the seal from the QC Dilution plate and discard the seal.
B. Transfer 10 µL of each QC Dilution Plate sample to the plate labeled OD.
This plate is known from here on as the OD plate.
C. Add 90 µL nuclease-free water to each well of the OD Plate and mix by pipetting up and down.
Change pipette tips after each addition.
Final sample mass dilution is 1:120.
See Appendix B, Sample Quantitation after Resuspension on page 225 for more information on
performing the Sample Quantitation.
4. Make Gel Samples:
A. Add 60 µL gel loading dye to each well of the Gel Sample Plate.
B. Transfer 3 µL of each QC Dilution Plate sample to the Gel Sample Plate.
C. Seal, vortex, and spin the plate.
5. Run gel as described in Appendix A, Fragmentation Quality Control Gel Protocol on page 223.
After the QC checks, the QC dilution plate, OD plate, and remaining gel samples can be discarded
once satisfactory results from the gel and OD 260 readings have been obtained.
At this point you can:
Proceed to Stage 4 — Denaturation and Hybridization, below; or
Store the Hyb Ready samples at –20 °C.
Stage 4 — Denaturation and Hybridization

You will proceed to Stage 4 in one of two ways:
Directly from Stage 3 without interruption.
With Hyb Ready samples that were stored at –20 °C after Stage 3.
To perform Stage 4:
If the Hyb Ready plate was stored at –20 °C, go to 1. Prepare Hyb Ready Samples Stored at -20 °C
on page 130
If you are proceeding directly from the end of Stage 4 — Denaturation and Hybridization on
page 128, go to 2: Prepare Equipment and Perform Denaturation on page 130
CAUTION: Parts of this stage should be performed under a fume hood.
Duration
Hands-on: 45 minutes including denaturation time
in GeneTitan MC: 23.5 to 24 hours Hyb Time
Required Input from Previous Stage

Hyb Ready plate
Equipment, Consumables, and Reagents Required

The following thermal cyclers are recommended:
Bio-Rad PTC-200/PTC-200G Thermal Cycler or
Whatman Biometra TRobot 96
BIO-RAD MJ 0240G
ABI 9700
ABI 2720
IMPORTANT: Always use the heated lid option when programming protocols.
The thermocycler needs to be programmed with the “Axiom Denature” protocol (see Thermal Cycler
Recommendations on page 97).
Table 4.19 Equipment Required for Stage 4 — Denaturation and Hybridization

Equipment Quantity
GeneTitan MC 1
Pipetaid 1
P200 12-channel Pipette 1
Pipette tips As needed
Thermal Cycler Appropriate thermal cycler, programmed with the 1

“Axiom Denature” protocol (see Thermal Cycler
Recommendations on page 97).
96 well metal chamber 1

warmed in a 48 °C oven*
* The metal chamber coming out of a 48 °C oven is warm to the touch. Gloves and mitts can be used if
it feels too hot.
Table 4.20 Consumables Required for Stage 4 — Denaturation and Hybridization

Consumable Vendor and Quantity
Part Number
Following components from Axiom Array Plate Kit: 1 kit includes:

One of the following Axiom Array plates: Affymetrix
Axiom Genome-Wide CEU 1 Array Plate (96 arrays) on protective P/N 901608 1
base
Axiom Genome-Wide ASI 1 Array (96 arrays) on protective base P/N 901640 1
Table 4.20 Consumables Required for Stage 4 — Denaturation and Hybridization

Part Number
Axiom™ myDesign™ Genotyping Array Plates, 1x96 format one 96- P/N 000780** one 96-array plate
array plate of one array type of one array type
array plate of two array types of one array type
array plate of four array type of one array type
**The Consumables for the GeneTitan MC instrument are packaged separately from the Axiom myDesign™
Genotyping Array Plates. The consumables are available in the GeneTitan Consumable kit (P/N 901606)
Hyb Tray P/N 500867 1
Table 4.21 Reagents required from the Axiom Reagent Kit

on Ice Temperature
Axiom Wash Buffer A

Module 3,
Room Temperature Axiom Wash Buffer B
96-Sample: P/N (both bottles; 1L)
901472
Axiom Water
1. Prepare Hyb Ready Samples Stored at -20 °C

To prepare Hyb Ready samples that were stored at -20 °C:
1. Warm up the Hyb Ready plate at room temperature for 5 minutes. It is not necessary to equilibrate
the plate for longer duration.
2. Make sure the Hyb Ready plate is sealed well.
If the plate is not sealed well:
A. Spin the plate and carefully remove the old seal.
B. If there is condensation on the top of the plate, blot dry gently with a Kimwipe.
C. Use a fresh seal and tightly reseal the plate.
3. Vortex the Hyb Ready plate briefly, then spin at 1000 rpm for 30 seconds.
4. Place the Hyb Ready plate at room temperature.
2: Prepare Equipment and Perform Denaturation

1. Preheat the 96-well metal chamber in a 48°C oven.
2. Warm up the array plate on the bench top before setting up hybridization on the GeneTitan MC.
A. Remove the array plate box from the 4 °C refrigerator where it is stored.
B. Open the box and remove the pouch containing the array plate and protective base.
WARNING: Do not remove the array plate from the protective base or touch the
surface of any arrays.
C. Leave the array plate in the pouch, unopened but placed on the bench for a minimum of 25 minutes
before opening and loading on the GeneTitan MC to allow the plate to come to room temperature.
D. At the end of the array warm up time, open the pouch and scan the array plate barcode into the
Batch Registration file (see Stage 1 — Create and Upload Batch Registration File on page 157).
3. Make sure the thermocycler is powered on and the Axiom Denature program with the heated lid
option has been selected.
4. Open the lid of the thermal cycler and place the sealed Hyb Ready plate on the thermal cycler.
5. Close the lid.
6. Start the Axiom Denature program, described on Thermal Cycler Recommendations on page 97).
7. While the program is running:
A. Prepare the reagents from Module 3 as described in Table 4.22:
Table 4.22 Reagents from Module 3 (P/N 901472)

Reagent Temp Out of Treatment
Module*
Axiom Wash Buffer A Room Temp Invert 2-3X for mixing before filling GT bottle
Axiom Wash Buffer B Room Temp Invert 2-3X for mixing before filling GT bottle
Axiom Water Room Temp N/A
Notes:
*Temp Out of Module: temperature the reagent is held at immediately after removal from
module
N/A: not applicable in this case
B. Set up the GeneTitan MC (see Setup the Instrument on page 158).

C. Upload the Batch Registration File (see Stage 1 — Create and Upload Batch Registration File on
page 157).
3: Prepare Hybridization Tray and Load into GeneTitan MC
CAUTION: It is recommended to perform the next set of steps under a fume hood.
1. After the Axiom Denature program has completed, remove the Hyb Ready plate from the
thermocycler and place into a 96-well metal chamber that has been pre-warmed in an oven at 48 °C.
2. Move the metal chamber containing the denatured Hyb Ready plate to a fume hood.
3. Remove Microamp seal from Hyb Ready plate and discard.
4. Remove the Hyb Tray (from Axiom Array Plate kit) from packaging.
5. Label the Hyb Tray. See the note below and Figure 4.6 on page 132 for more information.
IMPORTANT: It is critical that you write only on the proper location of the proper edge
of the Hyb tray. Do NOT write on any other side, as this can interfere with sensors inside
of the GeneTitan MC Instrument and result in experiment failure. To ensure proper
placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can
also mark the notched corner of the trays and lids.
You may label the Hyb Tray on the front part of the short side of the tray, next to the notch at the
left, as shown in Figure 4.6. The proper section for labeling is closest to the notched corner,
corresponding to the A1 and B1 wells.
Writing on the wrong side of the Hyb tray, or on the wrong part of the long side, may interfere with
the operation of sensors in the GeneTitan MC.
6. Place the Hyb Tray under the fume hood.
7. Using a P200 12 channel pipette, set at 105 µL, slowly transfer the denatured samples from the Hyb
Ready plate into the Hyb tray. Dispense to the first stop to avoid creating bubbles.
Change pipette tips after each transfer; discard the tip even if it shows some volume left.
There is no need to spread the sample around the bottom of the Hyb tray wells. Sample distribution
across the well will occur when the array plate is stacked together with the Hyb tray.
8. Load the array plate and Hyb tray into GeneTitan MC (see Load an Axiom Array Plates and Hyb Tray
Onto the GeneTitan MC on page 163).
IMPORTANT: The array plate must be loaded on the left side on its protective blue base,
as shown in the figure below. The clear plastic cover on top of the array plate SHOULD
NOT be loaded in the GeneTitan MC.
Load the Hyb tray on the right side without any covering. The hyb tray should not have
any bubbles.
IMPORTANT: After GeneTitan MC has stacked the array plate and hyb tray, the
instrument will extend the drawer. Manually check the stacking by gently pressing the six
latching points to confirm that the two parts are clamped properly, and check underneath
the arrays to make sure there are no bubbles. If bubbles are found, gently tap the plate
on top and the bubbles should disappear. Do NOT tip/tilt the array plate/hyb tray
sandwich while inspecting the bottom for bubbles. See Step 3 on page 166 for detailed
instructions.
Hybridization will continue on the GeneTitan for 23.5-24 hours before you can load the Ligation/
Staining/Stabilization reagent trays into the GeneTitan.
You must wait until the hybridization step on the GeneTitan is approximately 1.5 hours from completion
(22 hours after the start of hybridization) to begin Stage 5 — Manually Preparing Ligation, Staining, and
Stabilization Reagent Trays for the GeneTitan MC Instrument, below.
Long delays between sample denaturation and loading into the GeneTitan for hybridization should be
avoided. However, if denaturation has begun and the GeneTitan is found not to be ready for hybridization,
then:
If the Hyb Ready samples have not been transferred to the Hyb tray (still in the Hyb Ready plate),
the Hyb Ready plate should be held at 48 °C in the thermocycler until when the GeneTitan is ready,
at which point you should begin at Step 1 of 3: Prepare Hybridization Tray and Load into GeneTitan
MC on page 131.
If the samples have already been transferred to the hyb tray, the hyb tray should be sealed with plate
sealing film and placed in an oven at 48 °C until the GeneTitan is ready. Be sure to remove the plate
sealing film before loading into the GeneTitan.
Stage 5 — Manually Preparing Ligation, Staining, and Stabilization Reagent

Trays for the GeneTitan MC Instrument
This stage needs to be done when hybridization in the GeneTitan MC is near completion (1.5 hours before
completion), so the reagent trays can be loaded for the GeneTitan MC array processing steps.
Total time for this step: 1.5 hours, including reagent preparation, hands-on time and GeneTitan
Instrument loading.
IMPORTANT: The reagent trays prepared in this step, Stage 5 — Manually Preparing Ligation,
Staining, and Stabilization Reagent Trays for the GeneTitan MC Instrument are for the
continued processing of an Axiom Array Plate that
– has completed the hybridization stage.
– is ready for transfer to the fluidics area.
The reagent plates for the fluidics stage on the GeneTitan MC Instrument should not be
prepared in advance. Do not prepare these plates if there is no array plate ready for the
fluidics stage. Once prepared, these plates must be loaded onto the instrument as soon as
possible and should not be stored.
To prepare the reagent trays for GeneTitan MC:

1: Prepare the Reagents for Stage 5 on page 136
2: Prepare the Stain, Ligation and Stabilization Master Mixes on page 138
3: Aliquot Master Mixes and Axiom Hold Buffer into Trays on page 140
The following instructions are for manually preparing the reagents and trays required to process Axiom
array plates on the GeneTitan MC instrument. The reagents and trays required are as follows:
Table 4.23 Reagent trays required for the Axiom assay on the GeneTitan MC
Type of Tray Number of Trays Tray Designation Master Mix/Reagent
Stain tray with cover 2 S1 Stain 1 master mix
Stain tray with cover 1 S2 Stain 2 master mix
Stain tray with cover 1 Stbl Stabilization master mix
Stain tray with cover 1 Lig Ligation master mix
Scan tray 1 Scan Tray Hold Buffer
Table 4.24 Equipment Required for Stage 5 — Manually Preparing Ligation, Staining, and Stabilization Reagent Trays
for the GeneTitan MC Instrument
Equipment Quantity
GeneTitan MC 1
Ice bucket with ice 1
Microcentrifuge 1
Pipetaid 1
Table 4.24 Equipment Required for Stage 5 — Manually Preparing Ligation, Staining, and Stabilization Reagent Trays
for the GeneTitan MC Instrument
Equipment Quantity
Pipettes — single channel Pipettes — 12-channel: 1 each

P200 P200
P1000
Vortexer 1
Table 4.25 Consumables Required for Stage 5 — Manually Preparing Ligation, Staining, and Stabilization Reagent
Trays for the GeneTitan MC Instrument
Part Number
Aluminum foil (optional) As required
GeneTitan Consumables Kit Affymetrix 1 kit includes:

P/N 901606
Scan Tray P/N 501006 1
Stain Tray P/N 501025 5
Covers for trays P/N 202757 6
MicroAmp Clear Adhesive Film As required
Pipette, serological
5 x 1/10 mL (VWR P/N 53283-706) 1
10 x 1/10 mL (VWR P/N 53283-708) 2
Pipette tips As required for

pipettes listed in
Table 4.24
Solution basin, 100 mL sterile multichannel 5
15 mL Falcon tube Becton Dickinson 3

P/N 352097
50 mL Falcon tube 1
Reagents Required
Table 4.26 Axiom Reagents required for Stain and Ligation Stage
Module Reagent
Axiom Ligate Buffer
Axiom Ligate Enzyme
Module 4 Axiom Ligate Soln 1

Box 1 of 2
96-Sample: P/N 901278 Axiom Probe Mix 1
Axiom Stain Buffer
Table 4.26 Axiom Reagents required for Stain and Ligation Stage
Module Reagent
Axiom Ligate Soln 2
Axiom Probe Mix 2*
Axiom Wash A
Axiom Stain 1-A*
Module 4 Axiom Stain 1-B*

Box 2 of 2
96-Sample: P/N 901276 Axiom Stain 2-A*
Axiom Stain 2-B*
Axiom Stabilize Diluent
Axiom Water
Axiom Hold Buffer*
1: Prepare the Reagents for Stage 5

To prepare the reagents:
1. Prepare the reagents from Module 4, box 1 of 2 as described in Table 4.27:
Table 4.27 Reagents from Module 4, box 1 of 2 (96-sample: P/N 901278)

Reagent Temp Out of Treatment Storage before Master
Module* Mix
Axiom Ligate Buffer Thaw at Room Temp 1.Place on bench top at room temp for 30 min Place on ice
2.Examine for precipitate
3.Vortex twice
4.Examine for precipitate.
If any:
Warm bottle with your hands and vortex again
for thirty seconds
Axiom Ligate Enzyme Keep at –20 °C until Just before use: Place in –20 °C portable
ready to use 1.Flick 2 to 3 times to mix cooler
2.Spin.
3.Place in –20 °C portable cooler until use.
Axiom Ligate Soln 1 Thaw at Room Temp Vortex and Spin Place on Ice
Axiom Probe Mix 1 Thaw at Room Temp Vortex and Spin Place on Ice
Axiom Stain Buffer Thaw at Room Temp Vortex and Spin Place on Ice
Axiom Stabilize Soln Thaw at Room Temp Vortex and Spin Place on Ice
Notes:
Temp Out of Module*: temperature the reagent is held at immediately after removal from module
NOTE: The presence of some precipitate in Axiom Ligate Buffer is OK and will not adversely
impact assay performance. Follow the instructions above to resuspend any precipitate before
use.
2. Prepare the reagents from Module 4, box 2 of 2 as described in Table 4.28:
Table 4.28 Reagents from Module 4, box 2 of 2 (96-sample: P/N 901276)

Reagent Temp Out of Treatment Storage before Master Mix
Module*
Axiom Ligate Soln 2 Thaw at Room Vortex and Spin Store at Room Temp.
Temp (do not
place on ice!)
Axiom Probe Mix 2# Place on Ice Flick 2 to 3 times to mix, then spin Place on ice
Axiom Wash A Leave on bench 1.Vortex twice Place on bench top at room temp
2.Place on Bench for 30 min.
3.Look for precipitate.
4.Vortex again if necessary.
Axiom Stain 1-A# Place on ice Flick 2 to 3 times to mix, then spin Place on ice
Axiom Stain 1-B# Place on ice Flick 2 to 3 times to mix, then spin Place on ice
Axiom Stain 2-A# Place on ice Flick 2 to 3 times to mix, then spin Place on ice
Axiom Stain 2-B# Place on ice Flick 2 to 3 times to mix, then spin Place on ice
Axiom Stabilize Place on ice 1.Vortex and Spin Place on ice

Diluent 2.Look for precipitate
If any:
Warm tube to room temperature
and vortex again.
Axiom Water Place on ice N/A Place on ice
Axiom Hold Buffer# Room Temp Vortex Store at Room Temp away from light
Notes:
# These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.
* Temp Out of Module: temperature reagent is held at immediately after removal from module
NOTE: Occasionally, crystals are observed in Axiom Wash A and Axiom Stabilize Diluent upon
removal from 2-8 °C storage. Before using these solutions, the crystals should be dissolved by
warming the solutions to room temperature and then vortexing.
2: Prepare the Stain, Ligation and Stabilization Master Mixes

Label the Tubes and Solution Basins
To label the tubes:
1. Mark the side of each tube with one of designations shown in Table 4.29.
Table 4.29 Labeling master mix tubes for stain, ligation, and stabilization reagents
Conical Tube Number of Tubes Tube Designation Contents Place Tube:
50 mL 1 S1 Stain 1 Master Mix On ice
15 mL 1 S2 Stain 2 Master Mix On ice
15 mL 1 Stbl Stabilization Master On ice

Mix
15 mL 1 Lig Ligation Master Mix On ice
NOTE: Use a 5mL or 10 mL serological pipette to transfer Axiom Wash A, Axiom Water, and
Axiom Ligate Buffer. These bottles have narrow openings and a 25 mL serological pipette will
not fit.
2. Mark the side of each solution basin with one of the designations shown in Table 4.30.
Table 4.30 Labeling master mix tubes for stain, ligation, and stabilization reagents
Basin Designation Contents
S1 Stain 1 Master Mix
S2 Stain 2 Master Mix
Stbl Stabilization Master Mix
Lig Ligation Master Mix
Hold Axiom Hold Buffer
Prepare Stain 1 Master Mix

To prepare the Stain 1 Master Mix:
1. Use appropriate serological and single-channel pipettes to add reagents to the 50 mL tube labeled S1
in the order shown in Table 4.31. This recipe will provide enough for both S1 reagent trays.
Table 4.31 Stain 1 Master Mix

Reagent Per Array Master Mix 96+
To the tube marked S1, add:
Axiom Wash A 201.6 μL 22.2 mL
Axiom Stain Buffer 4.2 μL 463 μL
Axiom Stain 1-A 2.1 μL 231 μL
Axiom Stain 1-B 2.1 μL 231 μL
Total 210 μL 23.13 mL

(105 μL x 2)
2. Gently invert the tube 10 times to mix.

3. Place on ice and protect from direct light (e.g., cover with aluminum foil or ice bucket lid).
Prepare Stain 2 Master Mix

To prepare the Stain 2 Master Mix:
1. Use appropriate serological and single-channel pipettes to add reagents to the 15 mL tube labeled S2
in the order shown in Table 4.32.
Table 4.32 Stain 2 Master Mix

To the tube marked S2, add:
Axiom Wash A 100.8 μL 11.1 mL
Axiom Stain Buffer 2.1 μL 231 μL
Axiom Stain 2-A 1.05 μL 115.6 μL
Axiom Stain 2-B 1.05 μL 115.6 μL
2. Gently invert the S2 MM tube 10 times to mix.

Prepare Stabilization Master Mix

To prepare the Stabilization Master Mix:
1. Use appropriate serological and single-channel pipettes to add reagents to the 15 mL tube labeled Stbl
Table 4.33 Stabilization Master Mix

To the tube marked Stbl, add:
Axiom Water 93.19 μL 10.3 mL
Axiom Stabilize Diluent 10.50 μL 1.16 mL
Axiom Stabilize Soln 1.31 μL 144.8 μL
2. Vortex the master mix at high speed for 3 sec.

3. Place on ice.
Prepare Ligation Master Mix

The Ligation Master Mix is prepared in two stages.
Ligation Master Mix: Stage 1
To begin preparing the Ligation Master Mix:

1. Place the 15 mL conical tube marked Lig on ice.
2. Use appropriate serological and single-channel pipettes to add reagents to the 15 mL tube labeled Lig
Table 4.34 Ligation Master Mix Preparation — Stage 1

To the tube marked Lig, add:
Axiom Ligate Buffer 66.15 μL 7.3 mL
Axiom Ligate Soln 1 13.12 μL 1.45 mL
Axiom Ligate Soln 2 3.15 μL 348 μL
Sub–Total 82.42 μL 9.10 mL
3. Mix well by vortexing the tube for 3 seconds.

4. Place the tube marked Lig back on ice.
Ligation Master Mix: Stage 2
To finish preparing the Ligation Master Mix:

1. Remove the Axiom Ligation Enzyme from the –20 °C freezer and place in a cooler chilled to –20 °C.
2. Use appropriate serological and single-channel pipettes to add reagents to the 15 mL tube labeled Lig
Gently flick the Axiom Ligate Enzyme tube 2-3 times, then perform a quick spin immediately prior
to adding the enzyme to the Master Mix.
Table 4.35 Ligation Master Mix Preparation — Stage 2

Ligation Master Mix from Stage 1 82.42 μL 9.10 mL
Axiom Probe Mix 1 10.5 μL 1.16 mL
Axiom Probe Mix 2 10.5 μL 1.16 mL
Axiom Ligate Enzyme 1.58 μL 174.4 μL
3. Gently invert 10 times to mix (do not vortex).

3: Aliquot Master Mixes and Axiom Hold Buffer into Trays
Label the Trays

1. Gather the scan tray and the stain trays and covers from the Axiom Array Plate kit.
2. Label two Stain Trays S1
3. Label the remaining stain trays:

S2
Stbl
Lig
When preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument,
you will need to mark the front of each tray in a way that identifies its contents.
IMPORTANT: It is critical that you write only on the proper side of the front edge of Stain
Trays. The front edge of the tray is the short side with the lettering A through H. Do NOT
write on any other side, as this can interfere with sensors inside of the GeneTitan MC
Instrument and result in experiment failure. To ensure proper placement of lids onto stain
trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner
of the trays and lids.
You may label the Stain trays on the left side of the front of the tray as shown in Figure 4.3. The
correct side is closest to the notched corner, corresponding to the A1 through C1 wells.
Figure 4.7 Labeling GeneTitan stain trays
Notched corner of Labeling indicates this tray will

Stain Tray and Lid contain the Stain 1 master mix
Deionize Trays and Covers

Deionize the inside of each tray and cover now. Return the trays and covers to the bench top after
deionizing
See Appendix E, Deionization Procedure for GeneTitan Trays and Covers on page 241 for the
recommended technique.
About Aliquoting Reagents to Trays
IMPORTANT: Always aliquot reagents to the bottom of the tray. Avoid touching the sides or
the top of the wells with the pipette tips. Droplets close to or on the top of the well dividers
may cause the lid to stick to the tray during GeneTitan processing.
For all trays, pipette into trays on the bench top. If the trays are not being used immediately, protect them
from light by covering with foil or placing in a cabinet.
IMPORTANT: Remember to deionize the stain trays and the covers before preparing the stain
master-mixes
When aliquoting ligation, staining, and stabilization reagents to the trays, it is not necessary to spread the
reagent to each corner of the well. The reagent will spread evenly when the array plate is inserted into the
reagent tray during processing with GeneTitan MC.
Stain 1 Master Mix

To aliquot the Stain 1 Master Mix:
1. Pour the S1 Master Mix into the solution basin marked S1, placed on the bench top at room
temperature.
2. Using a P200 12 channel pipette with new pipette tips, aliquot 105 µL per well to both S1 trays —
dispense to the first stop only to avoid creating bubbles.
You do not need to change pipette tips between additions of the Stain 1 Master Mix.
3. If:
Bubbles are present, puncture them with a pipette tip.
Droplets of liquid splashed onto the well dividers, place a Kimwipe on top of the tray to blot and
remove. (Figure 4.8).
Figure 4.8 Well dividers in trays.
Example of a droplet of liquid

that has splashed onto the well
divider of a stain tray during
reagent aliquoting.
Ensure no droplets of liquid are
on top of the wells dividers. Blot
with a Kimwipe to remove.
4. Place covers on the S1 trays. Orient cover correctly on the tray with the notched corners
together (Figure 4.9).
IMPORTANT: Leaving liquid on the top of the dividers may cause excessive evaporation or
may form a seal that will restrict the removal of the GeneTitan tray cover.
Figure 4.9 Placing cover on stain tray
Notched corner of
Stain Tray and Lid
5. Protect the trays from light if not immediately loading onto the GeneTitan MC.
Stain 2 Master Mix

To aliquot the Stain 2 Master Mix:
1. Pour the Stain 2 master mix into the solution basin marked S2, placed on the bench top at room
temperature.
2. Using a P200 12 channel pipette with new pipette tips, aliquot 105 µL per well to the S2 tray —
dispense to the first stop.
You do not need to change pipette tips between additions of the Stain 2 Master Mix.
3. If:
remove.
4. Place a cover on the S2 tray. Orient the cover correctly on the tray with the notched corners
Notched corner of
Stain Tray and Lid
5. Protect the tray from light if not immediately loading onto the GeneTitan MC.
Stabilization Master Mix

To aliquot the Stabilization Master Mix:
1. Pour the Stabilization master mix into the solution basin marked Stbl, placed on the bench top at room
temperature.
2. Using a 12-channel P200 pipette with new pipette tips, aliquot 105 µL per well to the Stbl tray —
You do not need to change pipette tips between additions of the Stabilization Master Mix.
3. If:
Droplets of liquid splashed onto the well dividers, blot the top of the tray with a Kimwipe.
4. Place a cover on the tray. Orient cover correctly on the tray with the notched corners together.
Ligation Master Mix

To aliquot the Ligation Master Mix:
1. Pour the ligation master mix into the solution basin marked Lig. Keep the solution basin on ice.
2. Using a 12-channel P200 pipette with new pipette tips, aliquot 105 µL per well to the Lig tray —
You do not need to change pipette tips between additions of the Ligation Master Mix.
3. If:
remove.
4. Place a cover on the tray. Orient cover correctly on the tray with the notched corners
Notched corner of
Stain Tray and Lid
5. Protect the tray from light if not immediately loading onto the GeneTitan MC.
Axiom Hold Buffer

To aliquot the Axiom Hold Buffer to the Scan Tray:
1. Pour the Axiom Hold Buffer into the solution basin marked Hold, placed on the bench top at room
temperature.
2. Remove the Scan tray from its pouch.
3. Remove the scan tray cover, but leave the Scan Tray on its protective black base.
CAUTION: Do not remove the Scan Tray from its protective black base until loading onto
the GeneTitan MC instrument. To avoid scratching, do not touch the bottom of the tray
with pipette tips. Dispense hold buffer to the first stop only.
4. Prepare the barcoded Scan tray cover (PN 202757) that came with the scan tray by completing the
deionization procedure described in Deionization Procedure for GeneTitan Trays and Covers on
page 241. Place the cover as shown in Figure 4.13 on page 146 to prevent dust or static from
accumulating on the bottom of the cover.
IMPORTANT: If your scan tray came with a cover that does not match the picture of the scan
tray cover or if your scan tray cover does not have a barcode, then please contact your Field
Application Specialist for a replacement scan tray cover. All tray covers must have a machine
readable barcode.
Figure 4.12 Scan tray with the clear cover and protective base
Notched corners of cover, tray and base
Figure 4.13 Loading the Scan Tray with Axiom Hold Buffer
Leave the Scan Tray in its protective black base while loading with Axiom Hold Buffer.
Cover protective base
5. Use a 12-channel P200 pipette with new pipette tips to aliquot 150 µL to each well of a Scan tray —
dispense to the first stop and avoid touching the bottom of the tray.
You do not need to change pipette tips between additions of the Hold buffer.
IMPORTANT: The Hold buffer requires 150 μL per well.
6. If droplets of liquid splashed onto the well dividers, place a Kimwipe on top of the tray to blot and
remove.
7. Cover the tray by orienting the notched corner of the scan tray cover over the notched edge of the tray
and the flat side of the cover against the scan tray and leave on the bench top (no need to protect from
light (Figure 4.13)).
CAUTION: Do not remove the Scan Tray from its protective black base until loading onto
the GeneTitan MC instrument. To avoid scratching, do not touch the bottom of the tray
with pipette tips. Dispense hold buffer to the first stop only.
See Stage 3 — Ligate, Wash, Stain and Scan on page 172 for instructions on loading the reagent trays.

5
Axiom™ Genotyping Assay:
Array Processing with the GeneTitan® MC Instrument
The Axiom™ Genotyping Assay is designed for processing 96 samples at a time on Axiom™ Genome-
Wide and Custom myDesign™ Array Plates. The protocol is performed in two sets of steps:
Target Preparation, performed with either:
P
Automated target prep, performed with the Biomek FX Target Prep Express
See Chapter 3, Axiom™ Genotyping Assay: Target Preparation with Biomek FXP Target Prep
Instrument on page 15
Manual target prep, performed on the lab bench without advanced automation
See Chapter 4, Axiom Genotyping Assay: Manual Target Preparation on page 95

Array processing, performed on the GeneTitan Multi-Channel (MC) Instrument
This chapter includes instructions for Part 2: Array Processing. These instructions are presented as
follows:
Before Using the GeneTitan MC on page 149
Stage 1 — Create and Upload Batch Registration File on page 157
Stage 2 — Hybridization on page 158
Stage 3 — Ligate, Wash, Stain and Scan on page 172
Before Using the GeneTitan MC
Proper Tray Alignment and Loading

Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC. Each
plate, cover and tray has one notched corner. The notched corner of plates, trays, covers and bases must
be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside
each GeneTitan MC drawer (Figure 5.1 and Figure 5.2 on page 151).
TIP: Mark the notched corner of each plate, cover and tray with permanent marker to help
ensure proper alignment and loading onto the GeneTitan MC.
CAUTION: Take care not to damage the consumables or bend the blue cover posts or scan
tray posts.
NOTE: The instrument control software will display a warning if it detects a problem during
the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and DI Water
bottles should be replaced if the software displays such a warning. Refer to Appendix F,
GeneTitan® MC Instrument Care on page 247 for the message displayed to the user and the
procedure for replacing the filters.
Figure 5.1 Proper alignment and loading of plates, covers and trays in the GeneTitan MC.
IMPORTANT: Remove the

plastic protective shipping
tray cover.
Notched corner of Array Plate aligned

with notched corner of blue base.
Tip
Mark the notched corner of each plate,
cover and tray with permanent marker to
help ensure proper alignment and loading.
Plates and trays must be seated in this groove.
The notched corner of all plates, bases, and

covers and must be seated in this corner of
the drawer per the Tray Alignment guide.
Chapter 5 | Axiom™ Genotyping Assay: Array Processing with the GeneTitan® MC Instrument 151
Figure 5.2 Array Plate with protective blue base and the Hyb Tray aligned and properly loaded into drawer 6.
IMPORTANT: When you install the consumables, ensure that the fingers are retracted. Do
not lay the consumables on top of the drawer fingers - this indicates that the instrument
is not functioning correctly. Please notify your Field Service Engineer if the fingers do not
retract automatically. You should place the trays into the instrument drawers when a
drawer is fully extended by the instrument. The fingers are retracted when the drawer is
open and are extended when the drawer is closed in order to restrain the consumable.
Stain Trays and Covers
IMPORTANT: Always place the flat side of the cover against the Stain Tray.
Figure 5.3 Placement of covers on trays
Correct placement of cover on Stain Tray. Incorrect placement of cover on Stain Tray.

Proper labeling for hyb trays and reagent trays is described in:

CAUTION: Writing on the wrong side of the Hyb tray, or on the wrong part of the long side,
may interfere with the operation of sensors in the GeneTitan MC.

Figure 5.5 Labeling GeneTitan Stain Tray
E-mail and Telephone Notifications from the GeneTitan MC

We strongly recommend that you configure the Affymetrix GeneChip® Command Console (AGCC)
software to send you GeneTitan MC notifications. It is critical that you know when the instrument
requires your attention — either for sample handling or troubleshooting. Rapid notification can lessen the
risk of sample loss.
Notifications can be sent to e-mail addresses and telephones. Refer to the AGCC user manual for
instructions.
The types of notifications available will let you know when a process:
Starts
Completes
Aborts
Encounters an error
GeneTitan MC Lamp
The GeneTitan MC uses a xenon arc lamp system that is warranted for 500 hours to provide illumination
for imaging the array at two wavelengths. The xenon lamp has a limited lifetime and needs to be replaced
at regular intervals.
The GeneTitan Instrument Control software provides a timer that indicates the remaining useful life of
the bulb and notifies you when it requires replacement. It is important to adhere to the warnings specified
in the GeneTitan MC user guide.
Refer to the GeneTitan MC Instrument User Guide, P/N 08-0308, or Appendix F, GeneTitan® MC
Instrument Care on page 249 of this user guide for details on replacing the lamp.
Refer to the GeneTitan MC Instrument User Guide, P/N 08-0308, for the Lambda LS and Smart controller
system. The Lamp and the controller should NEVER be switched ON or OFF manually. The GeneTitan
MC instrument control software manages the lamp activity and will switch the lamp ON and OFF as
required. It takes 10 minutes to warm-up the lamp. In idle mode the lamp will remain ON for 2 hours
before it is automatically switched OFF and if there are no more plates being transferred from the fluidics
to the imaging station. This is by design and intended behavior. Please do not try to save the lamp life by
turning OFF the switch on the lamp.
NOTE: The power switch on the shutter box should be ON at all times. The OPEN/CLOSE
switch on the shutter box should be at AUTO position at all times.
Setup Options for Array Plate Processing

The processes (setup options) available for processing array plates are shown in Figure 5.6. A brief
description of each option is given below.
Figure 5.6 Setup options for processing array plates
Hyb-Wash-Scan
This setup option enables you to hybridize, wash-ligate-stain-fix, and scan an array plate on the
GeneTitan MC.
Hyb: the array plate is moved to the hybridization oven inside the instrument. Each denatured sample
in the Hyb Tray is hybridized to an array on the Array Plate.
Duration for 96 samples = 23.5 hr
Wash: samples on arrays are ligated, washed, stained and fixed.

Duration for 96 samples = ~ 5 hr
procedure for replacing the filters
Scan: The array plate is moved to the imaging device in the GeneTitan MC and each array is scanned.
Duration for 96 samples = ~ 7.5 hr
Hyb-Wash
If this setup option is selected, array plate processing will stop after the array has gone through fluidics
processing. Use this option if an array plate cannot be scanned on the same GeneTitan MC as the one used
for hybridization and fluidics processing.
If the array plate cannot be scanned immediately after the Hyb-Wash process is complete:
1. Wrap the array plate (in the scan tray with black protective base) in aluminum foil to protect from
light.
No lid is required. Do not invert the plate stack. If inverted, the Hold Buffer will spill out of the tray.
To prevent liquid spillage, try to keep the plate level when handling the plates. Do not touch the
bottom optical surface of the scan tray.
2. Store at 4 °C.
3. Scan the array plate within 3 days or less.
When ready to scan the array plate:

1. Keeping the plate protected from light, bring the plate to room temperature for ~ 20 min.
2. Remove the aluminum foil and load onto the GeneTitan MC
Wash-Scan
Use this option if:
You wish to bypass the Hybridization step and perform only the Wash/Stain and Scan steps.
Wash-Scan-Resume
Use this option if:
It was necessary to hybridize the array plate in an oven separate from the GeneTitan MC.
Fluidics processing has been interrupted (e.g., a power failure occurs at your facility).
Scan
Use this option:
To rescan an entire array plate or specific arrays on a plate that failed to scan for reasons such as
bubbles or gridding failure.
If you have hybridized and performed the fluidics processes off the GeneTitan MC.
Unload Plates
Use this option to unload plates and trays from the instrument when:
Array plate processing is complete.
Array plate processing has been aborted.
Aborting a Process
If necessary, you can abort the processing of one or more array plates. Instructions and an example are
shown below in Figure 5.7.
If the instrument aborts a process, you can retrieve the array plate and related consumables as described
in Figure 5.7. An instrument-initiated abort may occur:
Due to improper placement of plates
If the UPS detects a long power interruption, draining the UPS to 75% power.
Figure 5.7 Manually aborting an array plate.
To abort array plate processing:

1. Click the Stop button.
2. Select the array plate that you want to 1.
abort.
3. Click Abort.
4. Click Yes.
5. Wait until the status of the array plate
in the WorkFlow window changes
from AbortRequest… to Aborted 2.
(5A and 5B).
6. Once aborted, retrieve the array plate
and other related consumables by:
– Using Setup Option: Unload Plates
– Loading a new array plate.
Exception: If reagents are loading, abort

the plate using the Cancel button
displayed in the reagent load step.
3.
Note: If the gripper is required to

complete the Abort process, the plate will
remain in the "AbortRequest" state until
the gripper becomes available.
4.
5A.
5B.
Stage 1 — Create and Upload Batch Registration File

In the AGCC software, you must create and upload a Batch Registration file before you begin Stage 2 —
Hybridization on page 158 (example shown in Figure 5.8). This file contains information critical for:
Data file generation during scanning
Tracking the experimental results for each sample loaded onto an array plate
1. If you have not already created a batch registration file, create one now. (See Appendix D,
Registering Samples in Affymetrix GeneChip® Command Console on page 237 for detailed
instructions.)
2. In AGCC, select the array plate format (96 samples) and open a batch registration file template.
3. Scan the array plate barcode into the yellow barcode field.
4. Enter a unique name for each sample and any additional information.
5. Save the file.
6. Upload the file.
IMPORTANT: It is very important to create and upload a batch registration file with your
sample information prior to starting Stage 2 — Hybridization on page 158.
Figure 5.8 Example of a batch registration file for an array plate

Stage 2 — Hybridization
Reagents Required
Reagents Required
Table 5.1 Reagents required from the Axiom Reagent Kit

on Ice Temperature
Axiom Wash Buffer A

Module 3,
Room Axiom Wash Buffer B
Temperature (both bottles; 1L)
Axiom Water
An Axiom Genome-Wide CEU 1 array plate or an Axiom Genome-Wide and custom myDesign™
Array Plate is required for this step. Prior to inserting this plate into the GeneTitan for hybridization,
the array plate should be brought to room temperature as described on Step 2 on page 130.
A hybridization tray containing denatured samples (from Step 2 on page 88 in Chapter 3 or Step 8 on
page 132 in Chapter 4) is also required for this step. The denatured samples should be transferred to
the hyb tray only after the GeneTitan is ready for loading the hyb tray in the "Load an Array Plate and
Hyb Tray Onto the GeneTitan MC Instrument" step on page 121.
Setup the Instrument

To setup the instrument:
1. Launch AGCC Launcher and select AGCC GeneTitan Control (Figure 5.9).
The system initializes. After initialization, the System Status tab is selected and the status of the
Hybridization Oven is displayed at the bottom of the Log window. The status should read:
<Time of day> System Ready
IMPORTANT: Please do not close the scanner application by right-clicking on it and choosing
the "Close" option. This will cause the scanner application to exit abnormally and cause
undue delay in processing the next plate. The correct way to close the application is described
in Shutting Down the GeneTitan MC on page 182.
Figure 5.9 Launching AGCC and initializing the GeneTitan MC instrument.
System ready
Figure 5.10 System setup tab and the information displayed in this pane.
System Setup tab
Setup Option: The various options you can

choose for processing Axiom array plates.
Barcode: The array plate barcode. Can be

scanned or entered manually.
Protocol Name: The protocol that GeneTitan MC will

run. The list of protocols displayed is based on the first 6
digits of the array plate barcode. Only the protocols that
are valid for the type of array plate loaded are displayed.
Workflow Steps: This field
displays an overview of the user
actions required to process an
array plate based on the setup
option selected.
Status: This field displays the actions that must be

performed to prepare or unload the GeneTitan MC
for the setup option that has been selected.
After each action you are instructed to click the
Next button or to press the blinking blue
Confirmation button located on the GeneTitan
MC.
2. Select the System Setup tab (Figure 5.10).

3. Configure the software as follows:
A. Setup Option: Hyb-Wash-Scan
Other options available are described under Setup Options for Array Plate Processing on
page 154.
B. Click Next.
NOTE: If there is not enough disk space, a message is displayed.

Delete or move .dat files to another location to free up enough disk space for the data
that will be generated by eight Axiom Array Platess.
96 Axiom Array Plate requires ~ 80 GB
C. Plate Information:
Barcode: Scan or manually enter the Axiom Array Plates barcode and click Next.
The first six characters of the barcode identify the type of plate being loaded, the protocol
GeneTitan MC will use to process the plate, and the imaging device parameters required for this
type of plate.
550094 <barcode> = Affymetrix 96-array plate
Figure 5.11 Barcode error message
If this error message is displayed:

Ensure that the library files for the type of array plate you
are using are correctly installed.

Try manually entering the array plate barcode.
Library files must be installed prior to launching
GeneTitan. If a library file must be installed exit

GeneTitan, install libraries and relaunch GeneTitan.
Protocol Name: Select the protocol name and click Next.

The system reads the first 6 digits of the array plate barcode to determine which protocols can
be run for the type of array plate that has been loaded. Only valid protocols are displayed.
550094.protocol = for Affymetrix 96-array plate barcodes
4. Complete the remaining workflow steps as follows:
A. Refill bottles with buffer (Figure 5.12 on page 162)
1) Fill these bottles:
Wash A: fill with Axiom Wash Buffer A — keep at 2 L full
Wash B: fill with Axiom Wash Buffer B — Use all 600 mL of Wash B from the reagent kit
per Axiom plate. Fill to 1L mark when processing two plates on the same day.
Rinse: fill with Axiom Water — keep at l L full
f
IMPORTANT:
Always ensure that the GeneTitan bottles containing Wash A and Rinse are above
the 50% mark when setting up the system to process an Axiom HT array plate. All
600 mL of the Wash buffer B from the Axiom reagent kit should be emptied into the
GeneTitan Wash B bottle when setting up the system to process a plate. This ensures
that the GeneTitan Wash B bottle is filled to more than the requisite 35% of Wash B
bottle volume. Also, do not overfill the bottles. Fill Wash Buffer B and Water bottles
to the 1 L mark only. Wash A keep at 2 L. We strongly recommend refilling these
bottles every time you are prompted to do so.
If the volume in any of these bottles becomes too low during a run, a message is
displayed (see Chapter 9, Troubleshooting on page 217). However, even if you fill
the bottle at this time, the instrument may not be able to successfully complete the
step that was in progress.
Wash B — if you intend to load two array plates on the same day, fill the Wash B
bottle to the 1L mark (use both bottles from the Axiom Reagent Kit).
2) Empty the waste bottle.

3) Press the Confirmation button on GeneTitan MC to continue. A fluidics check is run (~ 1 min).
Figure 5.12 Example of the remaining workflow steps
Workflow step
Specific instructions for

each workflow step
B. Empty trash bin

1) Open the trash bin and empty.
If already empty, the trash bin remains locked and the Status pane reads “Trash bin is empty.”
2) Press the Confirmation button to continue.
C. Remove consumable trays and plates
1) Remove used trays and plates when drawers open.
If no consumables to remove, the Status window reads “Drawers are empty.”
2) Press the Confirmation button to continue.
D. Continue to Load an Axiom Array Plates and Hyb Tray Onto the GeneTitan MC on page 163.
Load an Axiom Array Plates and Hyb Tray Onto the GeneTitan MC
The System Layout pane indicates the position of the various trays in each drawer during a GeneTitan
MC run at maximum throughput. This pane does not change as plates are loaded or removed.
Figure 5.13 System layout — location of plates inside the GeneTitan MC
Drawer Numbers
1
2
3
4
5
6
To load an Axiom Array Plate and Hyb Tray onto GeneTitan MC:
1. When drawer 6 opens, load the Array Plate and Hyb Tray as follows:
A. Examine the wells of the Hyb Tray for bubbles; puncture any bubbles with a pipette tip.
IMPORTANT: Removing bubbles at this step greatly reduces the chance of bubbles
under the arrays when the Hyb Tray and the Axiom Array Plates are clamped. Bubbles
under an array can result in black spots on the array image.
B. Load the Hyb Tray without the cover on the right side of the drawer (Figure 5.15 on page 164).
The array plate must be loaded on its protective blue base, as shown in Figure 5.15 on page 164 below.
The clear plastic cover on top of the array plate SHOULD NOT be loaded in the GeneTitan MC. See
Figure 5.1 on page 150 for more details on the correct way of loading the array plate.
C. Remove the Array Plate and protective blue base from its package.
To avoid dust or other damage, leave the Array Plate packaged until ready to load onto the
GeneTitan MC (Figure 5.14).
Figure 5.14 Array Plate packaging

Figure 5.15 Array Plate with protective blue base and the Hyb Tray properly loaded into drawer 6.
D. Load the Array Plate with the protective blue base on the left side of the drawer (Figure 5.15).
CAUTION: The notched corner of each plate, cover and tray must be aligned. When
loading onto the GeneTitan MC, the notched edge plates, covers and trays must be
aligned as indicated by the Tray Alignment guide in the drawer (Figure 5.15 on
page 164).
The error message shown in may be displayed. Plate barcodes must face the internal
barcode reader (back of the drawer). Improper tray positioning can cause the
GeneTitan MC to crash, and can result in substantial damage to the instrument and loss
of samples.
E. Press the Confirmation button.
Figure 5.16 Barcode error message
When you load the array plate left side of the drawer: The internal bar code reader reads the barcode of
the array plate and compares it with the barcode and the plate type specified in the Barcode field and Plate
Type field on the Setup page. If the information is correct, the application allows you to proceed to the
next step. If the instrument is unable to read the barcode, it will push the tray out and will prompt
(Figure 5.16) you to load the correct plate with the proper orientation into the instrument (Figure 5.15).
Click OK to retry and check the loading of the array plate; or
Click Skip if the instrument has problems reading the barcode and after verifying that the trays have
been placed in the proper orientation.
IMPORTANT: Do not install a 3 plate stack of trays. Confirm that you have removed
the clear plastic shipping cover as shown in Figure 5.1 on page 150.
F. Select the arrays to scan (instructions in Figure 5.17).

By default, all arrays are selected.
Figure 5.17 Selecting which arrays to scan an array plate
Message in Status Window.
Default – all arrays are selected

Single array - click one box
Multiple arrays
Click one box
Hold down the Ctrl key
Click another box in the same column
Group of arrays:
Click one box
Hold down the Shift key
Left click and drag the mouse
2. Click Next, then click OK to begin processing the samples (Figure 5.18).
The Array Plate is placed on top of the Hyb Tray and clamped (now referred to as the plate stack).
Figure 5.18 Click OK to start processing the first Array Plate and Hyb Tray
Click OK to confirm that you wish to proceed with hybridization.

The plate stack is in the left position (the left side of the drawer).
The System Status window is automatically selected and brought to the front of the computer monitor.
3. When drawer 6 opens and the prompt in Figure 5.20 on page 167 is displayed:
A. Remove the plate stack and gently press the two plates together at each clamping point.
Listen for a clicking sound which indicates that the plates are now clamped. No clicking sound
indicates the plates are already clamped (See Figure 5.19 for an example of a array plate
hybridization plate sandwich).
Figure 5.19 Array plate hyb sandwich
B. Inspect the bottom of the plate stack for bubbles under the arrays — do NOT invert the plates.
C. If bubbles are present, gently tap the plate until the bubbles move out from under the arrays —
do NOT unclamp the plate stack.
D. Return the plate stack to the drawer, and press the Confirmation button to proceed.
The message in Figure 5.21 may be displayed again if plate orientation is incorrect or if the Hyb
Tray barcode cannot be read. Click OK to proceed.
Figure 5.20 Location of camping points on the Array Plate and Hyb Tray
Clamping points on Axiom Array Plates and Hyb
Notched Corners
Array plate
Hyb Tray
Figure 5.21 Verification message
Load a Second Axiom Array Plates and Hyb Tray Onto the GeneTitan MC
When You Can Load a Second Array Plate and Hyb Tray
Once processing begins, you have a specific period of time during which you can load another Axiom
Array Plates and Hyb Tray. This period of time is displayed above the Hyb Oven Status pane
(Figure 5.22). You cannot load another Hyb Tray before or after this period of time.
IMPORTANT: You must load the next Array Plate and Hyb Tray during the period of time
displayed above the Hyb Oven Status. You cannot load another Hyb Tray before or after this
period of time. You will have to wait until the current process is finished.
NOTE: While the first plate is in the oven, you can load another plate if the time spacing
requirement is met. This is to ensure that the second plate does not have to wait for system
resources in its workflow. The time spacing is roughly equal to the longer of the wash-stain
or scan time of the first plate.
Figure 5.22 Loading a second Hyb Tray and hybridization oven status information
Additional plates cannot be loaded before or after this

period of time while the instrument is operating.
In this figure, the system is currently available.
This pane displays the period of time during which
another Array Plate and Hyb Tray can be loaded.
Position of plate stack in the hybridization oven. Only 1

plate being processed in this figure. As such, position 2
is blank.
Position 1 - left side of oven
Position 2 - right side of oven
Green indicates the current oven temperature is within

the target temperature range.
Yellow indicates oven temperature outside of target
temperature range.
1. Select the System Setup tab.

2. Load an Axiom Array Plates and Hyb Tray in the same manner that you loaded the previous plate and
tray.
A. Scan or manually enter the Axiom Array Plates barcode, then click Next.
B. Load the Axiom Array Plates with the blue base and the Hyb Tray without the cover, then press
the Confirmation button.
C. Select the arrays to scan, then click Next.
D. Ensure that the plates are clamped securely when prompted, then press the Confirmation button.
E. Click OK when prompted to resume plate processing (Figure 5.23).
Figure 5.23 Confirm resume processing prompt
Select the System Status tab to view Axiom Array Plates status in the WorkFlow window (Figure 5.24).
Figure 5.24 Example of the WorkFlow window when two plates are loaded and are in the hybridization oven
Left and Right positions = the position of the Scan Tray in drawer 2 (left or right side of the drawer).
Status Window Prompts and Actions Required
Table 5.2 Refilling buffer bottles and emptying the waste bottle
Status Window Prompt Action Required Receptacle – Reagent
Buffer bottles have been depressurized. ** Replenish the fluid in Wash Bottles A and Wash Bottle A – fill with Axiom Wash
Please refill buffer into the bottles. B, and the Rinse bottle. Buffer A up to 2 L.
Empty the waste bottle. Empty the Waste Bottle. Wash Bottle B – fill with Axiom Wash
Press the Confirmation button to continue. Buffer B to the 1 L mark.
Rinse – fill with Axiom Water to the 1 L
mark.
Do not overfill these bottles.
** Every time you are prompted to refill the buffer bottles, the system runs a fluidics check (duration ~ 1 min).
Table 5.3 Emptying the trash bin

Status Window Prompt Action Required Receptacle – Reagent
Empty trash bin Open and empty the trash bin. —

Press the Confirmation button to continue.
NOTE: If the trash bin is empty, you will not be

able to open it. Continue the process by pressing
the blue confirmation button
Table 5.4 Loading the array plate and Hyb Tray; barcode error messages
Status Window Prompt Action Required Reagent – Receptacle
Load Array Plate Tray on [Left/ Load the array plate with the blue base and the Hyb Tray in Hyb Tray loaded with
Right] side of Drawer. Load drawer 6. denatured samples.
Hyb Tray without cover on IMPORTANT: The blue base must remain in “left side HTA in”
[Left/Right] side of Drawer. even when empty.
IMPORTANT: The trays must be positioned. If the trays are
placed incorrectly, the software will display an error dialog box

indicating the barcode could not be read.
Press the Confirmation button to continue.
Text version of the error message These messages are

Warning: The system was not able to verify the array plate barcode. displayed if:
Please verify that the tray on the left side of the drawer is a blue cover and if applicable, an array plate, in A plate has been
the correct ORIENTATION. The right side of the drawer should contain a hyb tray, if applicable, in the correct loaded improperly.
ORIENTATION. The bar code is missing
Details: or obscured
The consumable is either not the correct consumable, not loaded correctly, or its barcode is not readable.
Proceeding with an incorrect or incorrectly loaded consumable can result in a loss of consumables, loss of
samples and may require a field service engineer to service the instrument.
Refer to the System Setup Tab or the User Guide provided with the Assay or AGCC for instructions on
proper consumable placement.
Press the flashing blue confirmation button or...
Press OK, GeneTitan will verify the barcode and orientation.
Press Skip, GeneTitan will NOT verify the barcode and orientation. The barcode entered at registration will
be used.
Table 5.5 Selecting which arrays to scan

Status Window Prompt Action Required Reagent and Receptacle
Select arrays to scan Accept the default (all arrays selected) if —

appropriate. Otherwise, select the arrays to be
scanned.
Click Next, then click OK to start processing.
Stage 3 — Ligate, Wash, Stain and Scan
Scan Tray with Axiom Hold Buffer

Cover the tray by orienting the notched corner of the cover over the notched edge of the tray and leave
on the bench top (no need to protect from light; Figure 5.25).
CAUTION: Do not remove the Scan Tray from its protective black base. Leave the Scan
Tray in the base until loaded onto the GeneTitan MC. When handling the Scan Tray, the
bottom glass surface of the tray should not be touched.
Figure 5.25 The Scan Tray with cover on the black base.
Always leave the Scan Tray in its protective black base.

Proper Installation of the GeneTitan Tray Consumables.

It is very important that you install the GeneTitan tray consumables in the proper orientation. The barcode
faces into the instrument.
Figure 5.26 You must rotate and install the trays so that the barcode faces into the instrument.
Notch
(This faces out and left)
FRONT
(OF INSTRUMENT
FACING YOU)
Barcode
(This faces BACK TO
THE REAR of the instrument)
Turn the tray and cover combo so
that the barcode faces BACK AND
INTO the instrument and the notch
faces OUT AND TO THE LEFT.
Figure 5.27 The proper installation of the GeneTitan tray consumables (the image shows the Stain Tray and the Stain Tray
cover as an example)
Barcode faces
in and back.
FOR RESEARCH
USE ONLY
FOR RESEARCH
USE ONLY
Notch faces out and left.

“Affymetrix For Research Use Only”
faces out .
Load Trays onto the GeneTitan MC

To load trays onto the GeneTitan MC:
When hybridization of an Axiom Array Plates has finished, a message (Figure 5.28) will alert you to
resume the workflow setup. Press OK and the software takes you directly back to the System Setup tab.
Figure 5.28 The Resume Workflow Setup Message
This prompt to continue into reagent load (Figure 5.28) occurs when the hyb is complete. “Estimated
Time Remaining” displayed under “Hybridization Oven Status” may display a time remaining of 0 to 30
minutes when the prompt occurs.
GeneTitan will allow reagent load to take place after either.
the estimated time counts down to zero or
the actual real world hyb time (as indicated by the computer clock) indicates the hyb is complete.
NOTE: The time estimate displayed on some systems may lag due to high CPU utilization.
GeneTitan allows the workflow to synchronize with the system clock to compensate for this
situation during the final half hour of the hyb time estimate. When this prompt to resume
reagent loading is displayed to the user there is no need to wait for the estimated time to
count down to zero.
Follow the prompts displayed to continue with staining, ligation, fixing and scanning.
1. Follow the prompts in the Status window.
A. Wash Bottles A and B, and the Rinse Bottle — refill as necessary (the system will prime itself
again); Waste bottle — empty if necessary.
Wash bottle A — 2 L. Wash Bottle B and Rinse Bottle — fill to 1 L mark only.
B. Empty the trash bin.
C. Remove consumable trays and plates as instructed, except for the blue base.
Leave the blue array plate base in drawer 6 even though the base is empty.
2. Load consumable trays and plates as follows:
A. Follow the prompts in the Status window (load sequence and prompts in Table 5.6).
B. Once loaded, examine each cover for droplets of liquid.
C. If any liquid is present, remove the tray, clean the cover and top of the tray with Kimwipes, and
reload the tray.
CAUTION:
Orient trays as indicated by the guide inside the drawer. Improper orientation may
cause the run to fail.
Remove the protective black base from the Scan Tray immediately prior to loading
Figure 5.29 on page 176).
Examine each cover for droplets of liquid after loading. Liquid on the cover can result in
capillary phenomenon. As a result, the tray may stick to the cover and be lifted out of
place inside the instrument.
Table 5.6 Sequence for loading the trays with reagents

Loading Left Right
Sequence by
Drawer
Number
Note: If the software is unable to verify the barcode on the scan tray and the scan tray cover, the
software will display the following error message
2 Scan Tray with cover — do not load the protective black base
(left side of drawer as indicated in Status window)
3 Stain Tray with Stain 1 Ligation Tray

Table 5.6 Sequence for loading the trays with reagents

Loading Left Right
Sequence by
Drawer
Number
4 Stain Tray with Stain 2 Fix Tray
5 Stain Tray with Stain 1 Empty
Figure 5.29 Scan tray loaded in drawer 2.
Scan Tray with cover

loaded in drawer 2.
Do NOT load the protective black

base packaged with the Scan Tray.
IMPORTANT: When you load the plates, or trays, insert them under the tabs, or fingers, that
may protrude into the stage. Confirm that the tray is not resting on these fingers.
Figure 5.30 Stain 1 Tray and Ligation Tray loaded in drawer 3.
Stain 1 Tray and Ligation Tray

loaded in drawer 3
Figure 5.31 Stain 2 Tray and Fix Tray loaded in drawer 4.
Stain 2 Tray and Fix Tray loaded

in drawer 4
Figure 5.32 Stain 1 Tray loaded in drawer 5.
Stain 1 Tray loaded in drawer 5.
3. At the prompt shown in Figure 5.33, click Yes to load another Axiom Array Plates and Hyb Tray.
Figure 5.33 Prompt asking to load another plate. Right or left position determined by the position of Axiom Array Platess
already in the GeneTitan MC.
4. Follow the prompts and:

A. Setup Option: select Setup Another Run, then click Next.
B. Scan or manually enter the Axiom Array Plates barcode, then click Next.
C. Select a protocol, then click Next.
D. When drawer 6 opens:

1) Remove the blue cover from the previous Axiom Array Plates.
2) Load a new Axiom Array Plates and new blue base on the left; load a new Hyb Tray on the
right.
3) Press the Confirmation button.
E. Click OK when prompted (Figure 5.34).
Figure 5.34 Confirm Resume Processing message
F. When drawer 6 opens, confirm that the plate stack is securely clamped, then press the
Confirmation button.
When processing resumes:
1. The plate stack which has finished hybridization is moved from the Hyb oven to drawer 1 temporarily
and then moved to the unclamp station after step 2 (it remains clamped).
2. The plate stack in drawer 6 is moved to the Hyb oven.
3. The plate is moved to the unclamped station.
4. The plate stack in the unclamp area is unclamped and moved into the fluidics area.
NOTE: At the end of a Hyb-Wash-Scan run, all plate and tray covers and the fixing tray cover
should be in the trash.
Figure 5.35 is an example of how the System Status Workflow window will appear when three Axiom
Array Platess are being processed.
Figure 5.35 Example of the System Status window — three Axiom Array Platess are being processed.
Workflow indicates the

number of plates being
processed and where they are in
the instrument. In this example,
three Axiom Array Platess are
being processed: 2 in the Hyb
Oven and 1 in Fluidics.
Estimated Completion Time
is for the current process.
Estimated Time Remaining

for fluidics is adjusted as
necessary. Adjustments can be
due to process interruptions
such as a drawer being opened.
Step currently executing in

Fluidics.
Continuing the Workflow

Once a plate has gone through the fluidics stage of the process, it is moved to the imaging device.
When the scanning process begins, the window shown in Figure 5.36 is displayed. This window must
remain open while Axiom Array Platess are being scanned.
CAUTION:
The Scan Control window must remain open while Axiom Array Platess are being scanned.
Closing this window will halt the scanning process. You can minimize this window if
necessary without creating any interference to the imaging.
Do not manually, or through the AGCC transfer utility, move any data associated with the
current plate that is being processed/scanned. Transferring data will dramatically slow
scanning and may cause the computer to freeze.
Figure 5.36 Scan control window
This window must remain open

while scanning is in progress.
If you close this window,
scanning will stop and delay
sample processing.
Storing Hyb Trays for Rehybridization

Hyb Trays can be stored after processing for the purpose of rehybridization. Store Hyb Trays as directed
below. See also Appendix C, Rehybridization on page 235 for more information.
To store a Hyb Tray with processed samples:
1. After completion of the GeneTitan run, unload the Hyb Tray and tightly seal it with an adhesive film.
The plate must be well-sealed to prevent cross-contamination between samples.
2. Press the four corners and sides of the tray to ensure that there is no space between the seal and plate.
3. Store the Hyb Tray at –20 °C.
Shutting Down the GeneTitan MC

This procedure assumes that all of the Axiom Array Platess loaded onto the GeneTitan MC have been
processed.
WARNING: Do not attempt to shut down the GeneTitan MC while array plates are being
processed.
To shutdown the GeneTitan MC:

1. On the System Setup page, open the Setup Options drop-down menu and select Unload Plates.
2. Unload all of the consumables as prompted.
3. Power off the GeneTitan MC by opening Tools > Shutdown from the menu.
4. Exit the AGCC software if it does not close automatically.
NOTE: If the instrument is processing an array plate, the software will not allow you to shut
down the system.
6
Automated Target Preparation for Processing Eight Axiom
Array Plates Per Week
Using one Biomek FXP Target Prep Express (Biomek workstation) and one GeneTitan MC Instrument,
the Axiom™ assay (using the 96-array plate) can be run at a throughput of eight Axiom™ Array Plates per
five-day work week. This chapter includes tables that present the timing of the steps required to perform
this workflow per 5-day work week, eight hours per day.
IMPORTANT: Experienced users and careful timing are critical for the successful execution of
this workflow.
During the initial week of startup, all work is done on the Biomek FXP Target Prep Express. You will
process eight plates of genomic DNA samples. At the end of this week, you will have what are now
referred to as eight plates of hyb-ready target (target).
Subsequent weeks of the workflow involve the simultaneous processing of plates on the Biomek FXP
Target Prep Express and on the GeneTitan MC Instrument. Each week:
Eight plates of target from the previous week are denatured and transferred to the GeneTitan MC
Instrument for hybridization and array plate processing.
Eight new plates of target are prepared on the Biomek FXP Target Prep Express.
Plate Numbering Scheme

Target preparation on the Biomek FXP Target Prep Express: sample plates are numbered 1 through 8.
Target prepared the previous week is processed on the GeneTitan MC Instrument. The plates denatured
and loaded onto the GeneTitan are now referred to as plates A through H. Target prepared on the
Biomek FXP Target Prep Express can be run on the GeneTitan MC Instrument in random order.
Overview of the 8-plate Workflow on the Biomek FXP Target Prep Express
Table 6.1 Overview of the Biomek FXP Target Prep Express workflow
Day Activities Plates
1 Amplify eight plates of genomic DNA. 1 through 8
2 Fragment and precipitate three plates amplified on Day 1. 1, 5, 8

Freeze five plates of amplified gDNA for fragmentation later in the week. 2, 3, 4, 6, 7
3 Fragment and precipitate two more amplified plates. 2, 3

Centrifuge, dry, resuspend and QC the three plates precipitated on Day 2. 1, 5, 8
4 Fragment and precipitate the three remaining amplified plates. 4, 6, 7

Centrifuge and dry the two plates precipitated on Day 3. 2, 3
5 Resuspend and QC the two plates dried on Day 4. 2, 3

Centrifuge, dry, resuspend and QC the three plates precipitated on Day 4. 4, 6, 7
Table 6.2 Time required for target preparation on the Biomek FXP Target Prep Express
Steps on the Biomek FXP Target Prep Express Time Required
Amplification 30 min
Fragmentation 2 hr
Resuspension 45 min
Off-deck centrifugation and drying 75 min
Off-deck QC gel and OD 45 min
Denaturation only 30 min
Denaturation and GeneTitan reagent plate preparation 45 min
Transfer denatured samples to GeneTitan 15 min
Transfer reagent plates to the GeneTitan 15 min
Time Required includes setup.
Overview of the 8-plate Workflow on the GeneTitan MC Instrument
IMPORTANT: Maintaining consistent timing during the set up of the GeneTitan is critical to
containing the user interventions of the eight plate workflow within an eight hour work day.
Once one process begins late, there is little opportunity to catch up until the end of the
workflow.
Table 6.3 Overview of the GeneTitan MC Instrument 8 plate workflow

1 Hybridize two plates of denatured target. A and B
2 Hybridize two plates of denatured target. C and D

Load reagent plates for fluidics and imaging of plates loaded on day 1 A and B
3 Hybridize two plates of denatured target. E and F

Load reagent plates for fluidics and imaging of plates loaded on day 2 C and D
4 Hybridize two plates of denatured target. G and H

Load reagent plates for fluidics and imaging of plates loaded on day 3 E and F
5 Load reagent plates for fluidics and imaging of plates loaded on day 4 G and H
The hybridization time for the Axiom assay on the GeneTitan MC Instrument is 23.5 to 24 hr (Table 6.4).
This provides a 30 min window during which you are prompted by the instrument control software to load
the reagents required for washing and staining. We recommend that you begin loading the reagent plates
onto the GeneTitan at the mid-point of this 30 min window. As such, the wash procedures will begin 24
hr after the start of hybridization. If catch-up is required in the framework of the 8-plate workflow, begin
loading reagents at the beginning of this 30 min window (i.e., immediately after prompted by the
software).
Chapter 6 | Automated Target Preparation for Processing Eight Axiom Array Plates Per Week 185
Table 6.4 Time required for array plate processing on the GeneTitan MC Instrument
Steps on the GeneTitan MC Instrument Time Required
Hybridization of two plates 23.5 hr each plate
First plate loaded at 9:30 a.m.

Second plate loaded at 5:00 p.m.
Loading reagent plates that were prepared on the 15 min

Fluidics 5 hr each plate
Imaging 96 arrays: 7.5 hr
Thawing Frozen Plates of Amplified DNA

To thaw frozen plates of amplified DNA:
For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.
Initial Target Prep Week — Biomek FXP Target Prep Express
Initial Target Prep Week — Day 1

Amplify eight plates of gDNA samples.
IMPORTANT:
All amplifications should be set up on Day 1 to allow for a 22 to 24 hr amplification
incubation for each plate.
Begin thawing the amplification reagents, particularly the Axiom Amp Soln, 60 min prior to
the start of each reaction.
Table 6.5 Initial Target Prep Week — Day 1 Activities

Monday a.m. Monday p.m.
8 9 10 11 12 1 2 3 4 5
1
2
3
4
5
6
7
8

Activity Plate Number Instrument Approximate
Start Times
DNA Amplification 1 10:00 a.m.
DNA Amplification 4 Biomek FXP Target Prep Express 11:30 a.m.
DNA Amplification 5 12:00 p.m.

Fragment and precipitate plates 1, 5 and 8.
Freeze amplified plates 2, 3, 4, 6, and 7 after each plate has incubated for 23 hr.
IMPORTANT:
Plates 1, 5 and 8 are fragmented and precipitated on Day 2 without freezing to preserve a
23 hr amplification incubation.
Store all plates not fragmented and precipitated on Day 2 at –20 °C following 23 hr of
amplification reaction incubation.
Precipitation is carried out at –20 °C overnight.

Tuesday a.m. Tuesday p.m.
8 9 10 11 12 1 2 3 4 5
1 — Fragment/
Precipitate
s2
s3
s4
5 — Fragment/
Precipitate
s6
s7
8 — Fragment/
Precipitate
sX = Seal tightly and store at –20 °C.

Start Times
Fragment and precipitate 1 Biomek FXP Target Prep Express 9:30 a.m.
Freeze (–20 °C) 2 — 10:00 a.m.
Freeze (–20 °C) 3 — 10:30 a.m.
Freeze (–20 °C) 4 — 11:00 a.m.
Freeze (–20 °C) 6 — 12:30 p.m.
Freeze (–20 °C) 7 — 1:00 p.m.
Fragment and precipitate 8 Biomek FXP Target Prep Express 1:30 p.m.

Thaw plates 2 and 3 (see Thawing Frozen Plates of Amplified DNA on page 185).
Fragment and precipitate plates 2 and 3.
Centrifuge, dry, resuspend and QC plates 1, 5 and 8.
IMPORTANT:
Amplified plates that are frozen must be thawed and thoroughly mixed by following the
procedure under Thawing Frozen Plates of Amplified DNA on page 185.
After being centrifuged and dried, plates 1, 5 and 8 are sealed and placed in a 4 °C
refrigerator until further processing later the same day.
Prior to resuspension and QC, plates 1, 5 and 8 must be brought to room temperature (place
on bench top for 30 min).

Wednesday a.m. Wednesday p.m.
8 9 10 11 12 1 2 3 4 5
2 - RT
2 — Fragment/
Precipitate
1, 5, 8
Centrif/Dry
3 - RT
3 — Fragment/
Precipitate
1-RT 1-Resus 1-QC
5-RT 5-Resus 5-QC
8-RT 8-Resus 8-QC
RT = Bring plate to room temperature.
Centrif = centrifuge offline
Resus = resuspend
QC = fragmentation QC gel and OD quantitation

Start Times
Bring plate to room temperature (RT) 2 — 8:30 a.m.
Spin and dry 1, 5, 8 Plate centrifuge and oven 9:45 a.m.
Resuspension 1 Biomek FXP Target Prep Express, 1:30 p.m.

Start Times
Off-deck QC 1 Plate spectrophotometer, e-gel system 2:15 p.m.
Resuspension 5 Biomek FXP Target Prep Express 2:15 p.m.

Centrifuge and dry plates 2 and 3.
IMPORTANT:
After being centrifuged and dried, plates 2 and 3 are sealed and stored at –20 °C.

Thursday a.m. Thursday p.m.
8 9 10 11 12 1 2 3 4 5
4 - RT
4 — Fragment/
Precipitate
2, 3
Centrif/Dry
6 - RT
6 — Fragment/
Precipitate
7 - RT
7 — Fragment/
Precipitate
Centrif = centrifuge

Start Times
Thaw 4 — 8:30 a.m.
Centrifuge and dry 2, 3 Plate centrifuge and oven 9:45 a.m.
Thaw 6 — 10:30 a.m.
Thaw 7 — 12:30 p.m.

Centrifuge and dry plates 4, 6 and 7.
Resuspend and QC plates 2, 3, 4, 6 and 7.
IMPORTANT:
Plates 2 and 3 must be brought to room temperature for 90 min prior to resuspension.
After being centrifuged and dried, plates 4, 6 and 7 are sealed. Place plates 6 and 7 in a 4 °C
refrigerator until further processing later the same day. Plate 4 can be left on the bench top.
Prior to resuspension and QC, plates 6 and 7 must be brought to room temperature (place

Friday a.m. Friday p.m.
8 9 10 11 12 1 2 3 4 5
2 - RT
3 - RT
2-Resus 2-QC
4, 6, 7
Centrif/Dry
3-Resus 3-QC
4-Resus 4-QC
6-RT 6-Resus 6-QC
7-RT 7-Resus 7-QC
Resus = Resuspension

Start Times
Bring to room temperature 2 — 8:00 a.m.
Resuspension and QC 2 Biomek FXP Target Prep Express 9:30 a.m.
Resuspension and QC 3 10:15 a.m.
Resuspension and QC 6 1:15 p.m.
Simultaneous Eight-Plate Workflow

The tables on the following pages provide a breakdown of the timing involved to simultaneously:
GeneTitan MC Instrument: Process eight array plates per week with target prepared the previous week
on the Biomek FXP Target Prep Express.
Biomek FXP Target Prep Express: Prepare eight new plates of target for processing the following week
on the GeneTitan MC Instrument.
IMPORTANT: Timing is critical.

The Biomek FXP Target Prep Express will be in operation for a large percentage of each work
day.
Plates for target preparation on the Biomek FXP Target Prep Express are numbered
1 through 8.
Plates with target that are now ready for denaturation and transfer to the GeneTitan MC
Instrument for hybridization, fluidics processing, and imaging are lettered A through H.
Plates of target from the previous week (1–8) can be processed on the GeneTitan MC
Instrument in any order.
Eight Plate Workflow — Day 1
Biomek FXP Target Prep Express Activities

Denature two plates of target prepared the previous week (plates A and B).
Amplify eight new plates of genomic DNA (1 through 8)
GeneTitan MC Instrument Activities

Transfer denatured plates to the GeneTitan MC Instrument and begin hybridization (plates A and B).
IMPORTANT:
Plates 1 through 8 are referred to as A through H when denatured and loaded onto the
GeneTitan MC Instrument. Plates 1 through 8 can go onto the GeneTitan in any order.
All amplifications are set up on Day 1 to maintain a 22 to 24 hr amplification incubation for
each plate.
Begin thawing the amplification reagents, particularly the Axiom Amp Soln, 60 min prior to
the start of each reaction.
Table 6.15 Eight Plate Workflow — Day 1 Activities

Monday a.m. Monday p.m.
8 9 10 11 12 1 2 3 4 5
Den A Hyb A
1
2
3
4
5
6
7
8
Den B Hyb B
Den = denature
Den time period includes 15 min to transfer the denatured sample plate from the Biomek workstation to the GeneTitan.
Begin thawing the amplification reagents, particularly the Axiom Amp Soln, 60 min prior to the start of each reaction.

Start Times
Denature on the FX A Biomek FXP Target Prep Express 8:45 a.m.
Load onto the GT and hybridize A GeneTitan MC Instrument 9:30 a.m.
DNA Amplification 3 Biomek FXP Target Prep Express 11:00 a.m.
DNA Amplification 7 Biomek FXP Target Prep Express 1:30 p.m.
Denature on the FX B Biomek FXP Target Prep Express 4:15 p.m.
Load onto the GT and hybridize B GeneTitan MC Instrument 5:00 p.m.

Denature two plates of target from the previous week (plates C and D).
Prepare reagent plates for the GeneTitan MC Instrument (for plates A and B already on the GeneTitan).
Transfer the denatured samples and reagent plates to the GeneTitan MC Instrument.
Freeze amplified plates 2, 3, 4, 6, and 7 after each plate has incubated for 23 hr

Load reagent plates for A and B. These plates are moved from the hyb oven to the fluidics area. After
fluidics, the plates move to the imaging area of the instrument. Load plates C and D for hybridization.
IMPORTANT:
Plates 1, 5 and 8 are fragmented and precipitated without freezing to preserve a 23 hr
amplification incubation.
All plates not fragmented and precipitated on Day 2 should be stored at –20 °C following
23 hr of amplification reaction incubation.

Tuesday a.m. Tuesday p.m.
8 9 10 11 12 1 2 3 4 5
Thaw Den Hyb C

GT C
rgnts
End GT rgnt Fluidics and Scan A
Hyb A plates for A
1 — Fragment/
Precipitate
s2
s3
s4
5 — Fragment/
Precipitate
s6
s7
8 — Fragment/
Precipitate
Thaw Den Hyb
GT D D
rgnts
Hyb B GT rgnt **
23.5 - 24 hr plates for B B
** B = Fluidics and Scan B
Den = denature
Denature and GT rgnt plate time periods include 15 min to transfer the denatured plate and reagent plates from the Biomek to the
GeneTitan.
sX = Seal tightly and store at –20 °C.
GT rgnt plates = reagent plates for GeneTitan array plate processing.

Activity Plate Designation Instrument Approximate
Start Times
Thaw reagent for GT rgnt plate preparation A — 8:00 a.m.
Concurrently: Denature C Biomek FXP Target Prep Express 8:30 a.m.
Denature C Reagents for A
Prepare GT rgnt plates for A
Transfer to GeneTitan
Load GT rgnt plates and begin fluidics A to fluidics GeneTitan MC Instrument 9:30 a.m.
processing for A C to hyb oven
Load C and begin hybridization
Freeze (–20 °C) 2 — 10:00 a.m.
Freeze (–20 °C) 3 — 10:30 a.m.
Freeze (–20 °C) 4 — 11:00 a.m.
Freeze (–20 °C) 6 — 12:30 p.m.
Freeze (–20 °C) 7 — 1:00 p.m.
Thaw reagent for GT rgnt plate preparation B — 3:30 p.m.
Concurrently: Denature D Biomek FXP Target Prep Express 4:00 p.m.
Denature D Reagents for B
Prepare GT rgnt plates for B
Load GT rgnt plates and begin fluidics B to fluidics GeneTitan MC Instrument 5:00 p.m.
processing for B D to hyb oven
Load D and begin hybridization
Denature and GT rgnt plate preparation time periods include 15 min to transfer the denatured plate and reagent plates from the
Biomek to the GeneTitan.
Off-Deck Activities
Thaw plates 2 and 3.
QC — OD quantitation and fragmentation gel for plates 1, 5, 8.

Denature two plates of target from the previous week (plates E and F).
Prepare reagent plates for the GeneTitan MC Instrument (for plates C and D already on the GeneTitan).
Fragment and precipitate plates 2 and 3.
Centrifuge, dry, resuspend and QC plates 1, 5, 8.

Load reagent plates for plates C and D. These plates are moved from the hyb oven to the fluidics area.
After fluidics, the plates will move to the imaging area of the instrument. Also load plates E and F for
hybridization.

Wednesday a.m. Wednesday p.m.
8 9 10 11 12 1 2 3 4 5
Thaw Den E Hyb E

GT
rgnts
End GT rgnt Fluidics and Scan C
Hyb plates for C
C
2 – RT
2 — Fragment/
Precipitate
1, 5, 8
Centrif/Dry
3 - RT
3 — Fragment/
Precipitate
1-RT 1-Resus 1-QC
5-RT 5-Resus 5-QC
8-RT 8-Resus 8-QC
Thaw Den F Hyb
GT F
rgnts
Hyb D GT rgnt Fluidics and
plates for D Scan D
Resus = resuspend
GT rgnt plates = reagent plates for GeneTitan array plate processing.
Den time period includes 15 min to transfer the denatured sample plate and reagent plates from the Biomek to the GeneTitan.

Start Times
Thaw reagents for GT rgnt plate preparation C — 8:00 a.m.
Concurrently: Denature E Biomek FXP Target Prep Express 8:30 a.m.
Denature E Reagents for C
Prepare GT rgnt plates for C
Load GT rgnt plates and begin fluidics C to fluidics GeneTitan MC Instrument 9:30 a.m.
processing for C E to hyb oven
Load E and begin hybridization
Resuspension 1 Biomek FXP Target Prep Express, 1:30 p.m.
Thaw reagents for GT rgnt plate preparation F — 3:30 p.m.
Concurrently: Denature F Biomek FXP Target Prep Express 4:00 p.m.
Denature F Reagents for D
Prepare GT rgnt plates for D
Load GT rgnt plates and begin fluidics D to fluidics GeneTitan MC Instrument 5:00 p.m.
processing for D F to hyb oven
Load F and begin hybridization
Off-Deck Activities
Bring plates 4, 6 and 7 to room temperature.

Denature two plates of target from the previous week (plates G and H).
Prepare reagent plates for the GeneTitan MC Instrument (for plates E and F already on the GeneTitan).
Fragment and precipitate plates 4, 6, 7.
Centrifuge and dry plates 2 and 3.

Load reagent plates for E and F. These plates are moved from the hyb oven to the fluidics area. After
fluidics, the plates move to the imaging area of the instrument. Load plates G and H for hybridization.
IMPORTANT:
After being centrifuged and dried, plates 2 and 3 are sealed and stored at –20 °C.
Table 6.21 Eight Plate Workflow— Day 4 Activities

Thursday a.m. Thursday p.m.
8 9 10 11 12 1 2 3 4 5
Thaw Den G Hyb G

GT
rgnts
End GT rgnt Fluidics and Scan E
Hyb E plates for E
4 - RT
4 — Fragment/
Precipitate
2, 3
Centrif/Dry
6 - RT
6 — Fragment/
Precipitate
7 - RT
7 — Fragment/
Precipitate
Thaw Den H Hyb
GT H
rgnts
Hyb F GT rgnt Fluidics and
plates for F Scan F
Denature and GT rgnt plate preparation time periods include 15 min to transfer the denatured plate and reagent plates from the
Biomek to the GeneTitan.
Table 6.22 Eight Plate Workflow— Day 4 Activities

Activity Plate Instrument Approximate
Designation Start Times
Thaw reagents for GT rgnt plate preparation E — 8:00 a.m.
Concurrently: Denature D Biomek FXP Target Prep Express 8:30 a.m.
Denature G Reagent for E
Prepare GT rgnt plates for E
Load GT rgnt plates and begin fluidics E to fluidics GeneTitan MC Instrument 9:30 a.m.
processing for E G to hyb oven
Load G and begin hybridization
Spin and dry 2, 3 Plate centrifuge and oven 9:45 a.m.
Thaw reagents for GT rgnt plate preparation F — 3:30 p.m.
Concurrently: Denature H Biomek FXP Target Prep Express 4:00 p.m.
Denature H Reagents for F
Prepare GT rgnt plates for F
Load GT rgnt plates and begin fluidics F to fluidics GeneTitan MC Instrument 5:00 p.m.
processing for F H to hyb oven
Load H and begin hybridization
Off-Deck Activities
Bring plates 2 and 3 to room temperature.

Centrifuge and dry plates 4, 6, 7.
Resuspend and QC plates 2, 3, 4, 6, 7.

Load reagent plates for plates E and F. These plates are moved from the hyb oven to the fluidics area.
After fluidics, the plates will move to the imaging area of the instrument. Also load plates G and H for
hybridization.
IMPORTANT:
Plates 2 and 3 must be brought to room temperature for 90 min prior to resuspension.
After being centrifuged and dried, plates 4, 6 and 7 are sealed. Place plates 6 and 7 in a 4 °C
refrigerator until further processing later the same day. Plate 4 can be left on the bench top.
Prior to resuspension and QC, plates 6 and 7 must be brought to room temperature (place

Friday a.m. Friday p.m.
8 9 10 11 12 1 2 3 4 5
End GT rgnt Fluidics and Scan G

Hyb plates for G
G
2 - RT
3 - RT
2-Resus 2-QC
4, 6, 7
Centrif/Dry
3-Resus 3-QC
4-Resus 4-QC
6-RT 6-Resus 6-QC
7-RT 7-Resus 7-QC
Hyb H Rgnt Plate Fluidics and
Prep H Scan H

Resus = Resuspension
GT rgnt plate preparation time periods include 15 min to transfer the denatured plate and reagent plates from the Biomek to the
GeneTitan.

Start Times
Prepare GeneTitan reagent plates G Biomek FXP Target Prep Express 8:30 a.m.
Load reagent plates and begin G GeneTitan MC Instrument 9:30 a.m.
fluidics processing for G
Resuspension and QC 2 Biomek FXP Target Prep Express 9:30 a.m.
Prepare GeneTitan reagent plates H Biomek FXP Target Prep Express 4:00 p.m.
Load reagent plates and begin H GeneTitan MC Instrument 5:00 p.m.
fluidics processing for H

7
Automated Target Preparation for Processing Two Axiom Array
Plates per Week
The two array plate/week automated target prep workflow enables you to do the target preparation and
array processing for the Axiom Genome -Wide 96 Array in the same week.
This workflow is performed using one Biomek FXP Target Prep Express (Biomek workstation) (see
Chapter 3, Axiom™ Genotyping Assay: Target Preparation with Biomek FXP Target Prep Instrument on
page 15) and one GeneTitan MC Instrument (see Chapter 5, Axiom™ Genotyping Assay: Array
Processing with the GeneTitan® MC Instrument on page 149). This chapter includes tables that present
the timing of the steps required to perform this workflow per 5-day work week, eight hours per day.
this workflow.
The two plate per week automated workflow is described in the following sections:
Overview of the 2-plate Workflow for Automated Target Preparation
Thawing Frozen Plates of Amplified DNA on page 204
Target Prep and Array Processing on page 204
Overview of the 2-plate Workflow for Automated Target Preparation

The table below displays the timing and duration of the hands-on processing necessary for performing
the two-plate workflow by one person.
Figure 7.1 Two plate per week automated target prep workflow
Plate Day 1 Day 2 Day 3 Day 4 Day 5

AM PM AM PM AM PM AM PM AM PM
8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6
A
B
X
Notes
Begin Thawing required reagents for the process.
Begin warming Axiom array plate to room temperature
x Day 3 2:45 PM: Store Hyb Ready Plate B at –20 °C
Color Code
Amplification
Fragmentation and Precipitation
Centrifugation and Drying
Resuspension and Hybridization Mix Prep
QC
Sample Denature/load array plate and Hyb Plate in GeneTitan MC
GeneTitan MC reagent plates prep and loading
The two plates are referred to as plates A and B in the target prep and in the GeneTitan Array Processing.
In order to process two plates during a 40-hour week, the steps should be performed in the order and with
the timing described in this chapter.
Table 7.1 Daily steps for Automated Target Prep workflow for two plates/week
Day Activities
1 Amplify 2 plates of genomic DNA.
2 Fragment and precipitate two plates amplified on Day 1.
3 Centrifuge, dry, resuspend and QC Plate A.

Denature Plate A and load samples and Array Plate into GeneTitan MC
Centrifuge, dry, resuspend and QC Plate B.
Store Plate B at –20 °C
4 Denature Plate B and load samples and Array Plate into GeneTitan MC
Prepare GeneTitan Reagent plates for Plate A and load into GeneTitan MC
5 Prepare GeneTitan Reagent plates for Plate B and load into GeneTitan MC
Table 7.2 Time required for target preparation on the Biomek FXP Target Prep Express
Steps on the Biomek FXP Target Prep Express Time Required
Amplification 30 min
Fragmentation 2 hr
Resuspension 45 min
Off-deck centrifugation and drying 75 min
Off-deck QC gel and OD 45 min
Denaturation only 30 min
Transfer denatured samples to GeneTitan MC 15 min
Prepare and Load reagent plates to the GeneTitan MC 60 min
Time Required includes deck setup.

The automated two plate workflow described in this chapter does not require freezing the plates of DNA
following amplification. If plates of amplified DNA are frozen because of an interruption of this
workflow, you should thaw them prior to the fragmentation step on Day 2 as described in section 2. Thaw
and Prepare the Reagents and Sample Plate on page 45.
Target Prep and Array Processing

The tables in the sections below show the steps that need to be performed on each day of the workflow.
Two Plate/Week Workflow - Day 1
NOTE: Genomic DNA sample for amplification should be prepared as described in Genomic
DNA Preparation and Requirements on page 9. Affymetrix recommends that you carry out
genomic DNA preparation in a dedicated pre-amplification room, or area, with dedicated
consumables such as pipettes, tips, vortexes, etc.
Chapter 7 | Automated Target Preparation for Processing Two Axiom Array Plates per Week 205
IMPORTANT: Amplification preparation should take place in an Amplification Staging Room

or dedicated area such as biosafety hood with dedicated pipettes, tips, vortex, etc.
Figure 7.2 Two Plate/Week Automated Target Prep Workflow — Day 1 Activities
Day 1 a.m. Day 1 p.m.

8 9 10 11 12 1 2 3 4 5
Plate
A Amp
B Amp
Notes
Begin thawing reagents and materials for the process
Color Code
Amp Amplification (see Stage 1 — DNA Amplification on page 40)
Table 7.3 Two plate/week Auto Target Prep Workflow--Day 1

Time Topic
9:30 Prepare DNA amplification reagents for Plate A
10:30 Start DNA amplification Plate A
1:30 Prepare DNA amplification reagents for Plate B
2:30 Start DNA amplification Plate B
See Stage 1 — DNA Amplification on page 40 for more information on the protocol.
Two Plate/Week Workflow — Day 2
Figure 7.3 Two Plate/Week Workflow — Day 2 Activities

8 9 10 11 12 1 2 3 4 5
Plates
A Frag
B Frag
Notes
Begin thawing required reagents
Color Code
Frag Fragmentation and precipitation (see Stage 2 — Fragmentation and Purification on page 53)

Time Topic
9:30 Prepare Frag reagents for Plate A
10:00 Start Fragmentation Plate A
1:30 Prepare Frag reagents for Plate B
2:00 Start Fragmentation Plate B
Figure 7.4 Two Plate/Week Automated Target Prep Workflow — Day 3 Activities

8 9 10 11 12 1 2 3 4 5
Plates
A R QC Denat/
Cent/Dry R/HP QC hyb
B
Cent/Dry R/HP QC
X
Notes:
X Store Hyb Ready Plate B at –20 °C
Color Codes
Cent/Dry Centrifugation and Drying (see 5. Centrifuge and Dry Pellets on page 61)
R/HP Resuspension and Hyb Mix Prep (see Stage 3 — Resuspension and Hybridization Preparation on page 62)
QC Quality control checks
Denat/hyb Sample Denature/load array plate and Hyb Plate in GeneTitan MC (see Stage 4 — Preparation for the GeneTitan MC
Instrument on page 72)
Table 7.5 Two plate/week Automated Target Prep Workflow--Day 3

Time Topic
9:00 Start Centrifugation of Plate A
9:00 Prepare Hyb reagents for Plate A
9:40 Start Drying of Plate A
10:05 Start Resuspension and Hybridization Preparation Plate A
10:50 Run QC
11:30 Start Denature Sample for Plate A
12:00 Start Hyb-Wash-Scan run Plate A

Chapter 7 | Automated Target Preparation for Processing Two Axiom Array Plates per Week 207
Table 7.5 Two plate/week Automated Target Prep Workflow--Day 3

Time Topic
1:00 Start centrifugation of Plate B
1:00 Prepare Hyb reagents for Plate B
1:40 Start Drying of Plate B
2:05 Start Resuspension and Hybridization Preparation Plate B
2:50 Run QC and Put Plate B in Freezer (-20C)
IMPORTANT: The GeneTitan reagent plates for array processing cannot be loaded until the
array plate has finished hybridization, and they should not be prepared more than 1.5 hours
before hybridization will finish. The GeneTitan reagent plates cannot be prepared ahead of
time and stored.
Figure 7.5 Two plate/week Automated Target Prep Workflow--Day 4
Plates Day 4 a.m. Day 4 p.m.

8 9 10 11 12 1 2 3 4 5
A GT Reagent
Prep/Load
B Denat/
hyb
Notes
Color Codes
Denat/Hyb Sample Denature/load array plate and Hyb Plate in GeneTitan MC (see Stage 4 — Preparation for the GeneTitan MC
GT Reagent GeneTitan reagent trays prep and load (see Stage 4 — Preparation for the GeneTitan MC Instrument on page 72)
prep/load

Time Topic
10:00 Start Denature Sample for Plate B (See Stage 4 — Preparation for the GeneTitan MC Instrument
on page 72)
10:30 Start Hyb-Wash-Scan run for Plate B
10:30 Prepare Wash-Stain reagents for Plate A
11:00 Prepare GeneTitan reagent trays for Plate A (See Stage 4 — Preparation for the GeneTitan MC
12:00 Load GeneTitan reagent trays for Plate A

time and stored.
Figure 7.6 Two plate/week Automated Target Prep Workflow--Day 5

8 9 10 11 12 1 2 3 4 5
Plate
A
B GT Reagent
Prep/Load
Notes
Color Codes
GT Reagent GeneTitan reagent trays prep and load (see Stage 4 — Preparation for the GeneTitan MC Instrument on page 72)
prep/load

Time Topic
9:00 Prepare Wash-Stain reagents for Plate B
9:30 Prepare GeneTitan reagent trays for Plate B (See Stage 4 — Preparation for the GeneTitan MC
10:30 Load GeneTitan reagent trays for Plate B

8
Manual Target Preparation for Processing Three Axiom Array
Plates per Week
When using the manual target prep protocol, one person can process up to three Axiom Genome-Wide
96-Array Plates in one forty-hour work week.
This chapter describes the timing of the steps for each sample and array plate that are required to perform
this workflow.
this workflow.
The three plate per week workflow is described in the following sections:
Overview of the 3-plate Workflow for Manual Target Preparation
Thawing Frozen Plates of Amplified DNA on page 212
Manual Target Prep and Array Processing on page 212
Detailed instructions for the manual target prep protocol and the array plate processing are given in:
Chapter 4, Axiom Genotyping Assay: Manual Target Preparation on page 95
Chapter 5, Axiom™ Genotyping Assay: Array Processing with the GeneTitan® MC Instrument on
page 149
Overview of the 3-plate Workflow for Manual Target Preparation

The table below displays the timing and duration of the hands-on processing necessary for performing
the three plate workflow by one person.
Figure 8.1 Three plate per week manual target prep workflow
Plate Day 1 Day 2 Day 3 Day 4 Day 5

AM PM AM PM AM PM AM PM AM PM
8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6
A
z
B
C
x y z
Notes
Begin Thawing required reagents for the process.
x Day 2 11 AM: Freeze Plate C
y Day 3 Noon: start plate C thawing
z Day 4 5:00 PM: Coupled operations on GeneTitan MC: Load reagents for plate A and Hyb tray and array plate for Plate C
Color Code
Amplification
Fragmentation and Precipitation
Centrifugation, Drying, Resuspension, Hybridization Mix Prep, and QC
Sample Denature/load array plate and Hyb Plate in GeneTitan MC
GeneTitan MC reagent plates prep and loading
The three plates are referred to as plates A, B, and C in the manual target prep and in the GeneTitan Array
Processing.
In order to process three plates during a 40-hour week, the steps must be performed in the order and with
the timing described in this chapter.
Table 8.1 Daily steps for Manual Target Prep workflow

1 Amplify 3 plates of genomic DNA. A, B, and C
2 Fragment and precipitate two plates amplified on Day 1. A, B

Freeze one plate of amplified DNA for fragmentation later in the week. C
3 Fragment and precipitate one plate. C

Centrifuge, dry, resuspend and QC two plates precipitated on Day 2. A, B
Denature and begin hybridization for one plate on GeneTitan A
4 Centrifuge, dry, resuspend and QC plates precipitated on Day 3 C

Denature and begin Hybridization for two plates on GeneTitan B, C
GeneTitan reagent plates preparation and loading A
5 GeneTitan reagent plates preparation and loading B, C
The timing of these steps is critical because of constraints on both the target preparation, done on the lab
bench, and the array processing, done using GeneTitan MC.
These constraints are described in more detail in:
Timing Issues for Manual Target Preparation on page 210
Timing Issues for GeneTitan MC Array Processing on page 211
Timing Issues for Manual Target Preparation

The GeneTitan reagent plates for array processing cannot be loaded until the array plate has finished
hybridization, and they should not be prepared more than 1.5 hours before hybridization will finish. The
GeneTitan reagent plates cannot be prepared ahead of time and stored.
Table 8.2 Time required for manual target preparation

Manual Preparation Hands-on Time Total Prep Incubation/Hybridization/
Required Time* Processing
Amplification 0.5 hr 1.5 hr 23±1 hr
Fragmentation and Precipitation prep 2 hr 2 hr Overnight Precipitation
Centrifugation/Drying/Resuspension 1 hr 20 min 2 hr N/A
QC gel and OD 30 min 30 min N/A
Denaturation and hyb tray/array plate 25 min 45 23.5-24 hr hybridization

loading on GeneTitan
GeneTitan reagent plate preparation and 1 hr 1.5 hr Additional time for processing:
loading on GeneTitan 96 arrays: 12.5 hr
* Total Prep Time includes reagent thawing time and hands-on time
Chapter 8 | Manual Target Preparation for Processing Three Axiom Array Plates per Week 211
Timing Issues for GeneTitan MC Array Processing
IMPORTANT: Maintaining consistent timing during the set up of the GeneTitan is critical to
containing the user interventions of the three plate workflow within a work day. Once one
process begins late, there is little opportunity to catch up until the end of the workflow.
The hybridization time for the Axiom assay on the GeneTitan MC Instrument is 23.5 to 24 hr (Table 8.3).
This provides a 30 min window during which you are prompted by the instrument control software to load
the reagents required for washing and staining.
Table 8.3 Time required for array plate processing on the GeneTitan MC Instrument
Steps on the GeneTitan MC Instrument Time Required
Hybridization of two plates in one day 23.5 hr each plate

First plate loaded at 9:30 a.m.
Second plate loaded at 5:00 p.m.
Loading reagent plates 15 min
Fluidics 5 hr each plate
Imaging 96 arrays: 7.5 hr
Changing Oven Temperatures for the Three Plate Workflow

Multiple ovens are required for manual target preparation. If you are running the three plate /week
workflow, you will need three ovens. Table 8.4 lists the different temperatures required for each step.
Table 8.5 provides a list of suggested settings for three ovens when performing the three plate/week
workflow.
Table 8.4 Oven temperatures needed for each step of the workflow
Workflow step Oven Temp
Amplification 30 °C
Stopping Amplification 65 °C
Pre-Fragmentation Incubation 37 °C
Fragmentation Incubation 37 °C
Drying 37 °C
Hybridization 48 °C
Table 8.5 Suggested settings for ovens when performing three plate/week manual target prep workflow
Day of Workflow Oven 1 Oven 2 Oven 3
Day 1 30 °C N/A N/A
Day 2 30 °C 65 °C 37 °C
Day 3 48 °C 65 °C 37 °C
Day 4 48 °C 65 °C 37 °C
Day 5 N/A N/A N/A


To thaw frozen plates of amplified DNA:
For example, pour Millipore water into a small tray. Place the frozen plate in the water in the tray.
Manual Target Prep and Array Processing
Manual Target Prep Workflow — Day 1

On this day you start amplification of the three plates: each plate must incubate 23±1 hours after
amplification begins.
All amplifications should be set up on Day 1 to allow for a 23±1 hr amplification incubation for each
plate and to minimize movement between pre-amplification and post-amplification areas.
Begin thawing the amplification reagents, particularly the Axiom Amp Soln, 60 min prior to the start of
each reaction.
IMPORTANT: Amplification preparation should take place in an Amplification Staging Room

or dedicated area such as biosafety hood with dedicated pipettes, tips, vortex, etc. See
Amplification Staging Room on page 96 for more information.
Figure 8.2 Manual Target Preparation Workflow — Day 1 Activities

8 9 10 11 12 1 2 3 4 5
Plate
A Amp
B Amp
C Amp
Notes
Begin thawing reagents and materials for the process
Color Code
Amp Amplification (see Stage 1 — DNA Amplification on page 107)
Table 8.6 Manual Target Prep Workflow — Day 1 Activities

Activity Plate ID Approximate
Start Times*
DNA Amplification A 9:30 a.m.
DNA Amplification C 10:30 a.m.
DNA Amplification B 1:30 p.m.
*Approximate start time indicates start of thawing of reagents
See Stage 1 — DNA Amplification on page 107 for more information on the protocol.

The tables below show the steps that need to be performed on the second day.
Plates A and B are fragmented and precipitated on Day 2 without freezing to preserve a 23 hr
amplification incubation.
IMPORTANT: Store plate C immediately after the end of the Amplification reaction without
stopping the Amplification reaction, at –20 °C following 23 hr of amplification reaction
incubation.

8 9 10 11 12 1 2 3 4 5
Plates
A Frag
B Frag
C x
Notes
x Freeze plate C
Color Code
Frag Fragmentation and precipitation (see Stage 2 — Fragmentation and Precipitation on page 115)

Start Times
Fragment and precipitate A 10:00 a.m.
Freeze (–20 °C) C 11:00 a.m.
Fragment and precipitate B 2:00 p.m.


Centrifuge, dry, resuspend and QC plates A and B.
Thaw plate C (see Thawing Frozen Plates of Amplified DNA on page 212).
Fragment and precipitate plate C.
Perform Denaturation on plate A.
Transfer plate A samples to Hyb Tray A
Load Hyb Tray A and Array Plate into GeneTitan MC and begin hybridization.
WARNING: The Hybridization Tray prep should take place under a running fume hood.
IMPORTANT: Amplified plates that are frozen must be thawed and thoroughly mixed by
following the procedure under Thawing Frozen Plates of Amplified DNA on page 212.

8 9 10 11 12 1 2 3 4 5
Plates
A C/D/R/QC Denat/
Hyb
B C/D/R/QC
C y Frag
Notes:
y Begin thawing plate C
Color Codes
Frag Fragmentation and Precipitation (see Stage 2 — Fragmentation and Precipitation on page 115)
C/D/R/QC Centrifugation, Drying, Resuspension, Hyb Cocktail Prep, and QC (see Stage 3 — Drying, Resuspension and QC on page 122)
Denat/hyb Sample Denature/load array plate and Hyb Plate in GeneTitan MC (see Stage 4 — Denaturation and Hybridization on
page 128)

Start Times
Centrifuge/Dry/Resuspend/QC A, B 9:00 a.m.
Thaw Plate C C 12:00 a.m.
Fragment and precipitate C 1:00 p.m.
Denature and Hyb A 4:00 p.m.

Denaturation of Samples/Load array plate and Hyb Plate in GeneTitan MC for plates B and C
Centrifuge, dry, resuspend, and QC plate C
GeneTitan reagent plates prep and loading for plate A
WARNING: The Hybridization Tray prep should take place under a running fume hood.
time and stored.
Plates Day 4 a.m. Day 4 p.m.

8 9 10 11 12 1 2 3 4 5
A GT Reagent z
prep/load
B Denat/
Hyb
C C/D/R/QC Denat/
Hyb z
Notes
z Coupled operations on GeneTitan MC: Load reagent trays for plate A and Hyb Tray/Array Plate for Plate C
Color Codes
C/D/R/QC Centrifugation, Drying, Resuspension, Hyb Cocktail Prep, and QC (see Stage 3 — Drying, Resuspension and QC on
page 122)
Denat/Hyb Sample Denature/load array plate and Hyb Plate in GeneTitan MC (see Stage 4 — Denaturation and Hybridization on
page 128)
GT Reagent GeneTitan reagent trays prep and load (see Stage 5 — Manually Preparing Ligation, Staining, and Stabilization
prep/load Reagent Trays for the GeneTitan MC Instrument on page 134)
Table 8.9 Manual Target Prep Workflow —Day 4 Activities

Start Times
Denature and Hyb B 8:45 a.m.
Centrifugation/Drying/Resuspension/QC C 9:30 a.m.
GeneTitan reagent prep and loading A 3:30 p.m.
Denature and Hyb C 4:15 p.m.

GeneTitan reagents prep and loading for plates B and C.
time and stored.

8 9 10 11 12 1 2 3 4 5
Plate
A
B GT Reagent
prep/load
C GT Reagent
prep/load
Notes
Color Codes
GT Reagent GeneTitan reagent trays prep and load (see Stage 5 — Manually Preparing Ligation, Staining, and Stabilization Reagent
prep/load
Trays for the GeneTitan MC Instrument on page 134)

Start Times
GeneTitan reagent tray prep and loading B 8:00 a.m.
GeneTitan reagent tray prep and loading C 3:30 p.m.
9
Troubleshooting
If a hardware problem is encountered while running the Axiom target preparation methods on the Biomek
FXP Target Prep Express, you can do the following:
Refer to these documents:
®
Biomek Liquid Handler User’s Manual, Beckman Coulter P/N 987834
®
Biomek Software User’s Manual, Beckman Coulter P/N 987835
For information on recovering a run, contact your Affymetrix Field Application Specialist.
For additional information on Biomek FXP Target Prep Express hardware, error messages, or to request
service, contact Beckman Coulter. Be sure to have the serial number of your workstation available.
GeneTitan Multichannel Instrument

Refer to the GeneTitan® Multi-Channel Instrument User’s Manual, P/N 08-0306 for further
troubleshooting information.
Table 9.1 GeneTitan Multichannel Instrument troubleshooting guidelines for the Axiom assay
Problem Possible Causes Possible Actions
Plate trapped in GeneTitan Plate (or plate with lid) not properly 1 Restart the GeneTitan Multichannel Instrument.
Multichannel Instrument. loaded in drawer. 2 Run the setup option Unload Plates
Cut edge of lid and plate not aligned. 3 If the plate remains trapped in the instrument,
Gripper failed to retrieve plate. call Affymetrix support.
System requires adjustment.
Computer frozen. Too many processes running Restart the computer and unload all of the plates.
Attempting to transfer data while an Plates in Hyb station: finish hybridization off-line.
array plate is being scanned (imaged). Plate in Scanner: rescan using Scan Only function
Plate in Fluidics: use Wash/Scan Resume to resume
the fluidics process

Do not manually, or through the AGCC
transfer utility, move any data associated
with the current plate that is being processed/
scanned.
Hybridization aborted: System-initiated abort: Array plate and Hyb Tray are still clamped:
System-initiated abort Power loss Contact your local field service engineer with
User-initiated abort information on the workstation model

The plate stack is moved to drawer 1.
Remove the plate stack and finish hybridization
offline.
Return the hybridized array plate to the
GeneTitan Multichannel Instrument and finish

processing using the Wash/Scan process.
FAILED messages See Failed Messages on page 219
FLUIDIC DIAGNOSTIC messages See Fluidic Diagnostic Messages on page 219.
Fluidics aborted: System-initiated abort: Follow the recommendations and instructions

System-initiated abort Power loss under Wash/Scan Resume on page 221.
User-initiated abort
User-initiated abort:
Incorrect protocol selected
Miscellaneous Messages
Table 9.2 Miscellaneous messages and recommended actions

Message and Recommended Action
Recommendation: click Yes.

If you click No, nothing will occur. Homing will not
complete and you not be able to use the system.
The item held by the gripper will be moved to either:
Drawer 2 — plates and trays
Trash Bin — covers

Indicates that an item is in the gripper, and normal startup of the
The drawer names will reflect the location (left or Right)
GeneTitan Multichannel Instrument is not possible. The item must be
and the drawer number (1 through 6).
removed from the instrument before you can begin processing array
plates. Examples:
Drawer2L_Hta_DOWN = Scan tray on left side of drawer 2
HtaHyb = Clamped Hyb Tray and Array Plate
Drawer(n)L/R_Hta_DOWN where n is the drawer number
and L or R to indicate the left or right side.
The _Hta_ (second term) indicates the item held. An
example is drawer1R_HtaHyb_DOWN indicating it is an
array plate with a hyb tray or
Drawer2L_ScanHta_Pk_DOWN indicating it is an array
plate with a scan tray
The drawer listed in the message is not fully closed. Manually push the drawer back into the instrument until it is fully closed. There
are two stop positions with audible clicks; push until you hear the second click and the drawer is fully seated.
Check that the array plate barcode has been entered

correctly.
Ensure that the library files required for the type of array
plate you are using have been installed, and are installed
in the correct directory.
Restart the GeneTitan instrument control software after
library files have been installed.
Chapter 9 | Troubleshooting 219
Failed Messages
Table 9.3
Problem and Possible Causes
If this message is displayed:

during a water wash step, array processing has been
compromised.
during cleanup, array processing is OK, but cleanup will
not be complete.
Always ensure that the GeneTitan bottles containing
Rinse bottle — fluid level too low or bottle empty. Wash A and Rinse are above the 50% mark when setting
up the system to process an Axiom HT array plate.
All 600 mL of the Wash buffer B from the Axiom reagent
kit should be emptied into the GeneTitan Wash B bottle
when setting up the system to process a plate. This ensures
that the GeneTitan Wash B bottle is filled to more than the
requisite 35% of Wash B bottle volume.
Fluidic Diagnostic Messages
Table 9.4 Problem messages

About this message:

BUFFERX = Buffer bottle A, B or Rinse
WASHX = Wash A or B reservoir in the fluidics station.
Recommended actions:
Replenish fluid level in the Rinse or Wash Bottle B to the
1L mark. Do not overfill.
Only replenish bottles when prompted by the UI.
Replenishing during fluidic processing may cause

system malfunction including overflowing inside the
system and more problems. The only thing to do while
a plate is running is to make sure bottle caps are
secure.
Replenish fluid level in Wash Bottle A to 2 L.
Secure the bottle cap.
Replace the filter
Instructions for filter replacement in the GeneTitan®

Multi-Channel Instrument User’s Manual, P/N 08-0306.
If the problem persists, call Affymetrix support.
The typical cause is an unsecure bottle cap.
If the failure is detected during priming, the instrument

will pause and wait for the problem to be corrected.
If the failure is detected during another process, and if the

cause is a clogged filter, wait until the end of the run to
replace the filter.
Instructions for filter replacement in the GeneTitan®
Multi-Channel Instrument User’s Manual, P/N 08-0306.
Table 9.4 Problem messages

When the instrument experiences a loss in Clean Dry Air (CDA) Possible Causes
pressure, the software will display the warning message. Please verify that the facility CDA or the portable CDA
compressor is in working condition. Refer to the
GeneTitan MC site prep guide for the portable compressor
model that has been validated with the GeneTitan MC
instrument.
Contact your local field application specialist and notify
the engineer about the error message.
When the pressure is detected again, a dialog message confirming the

availability of CDA pressure is displayed.
Filter Error Messages The filters in the GeneTitan fluidics bottles (Wash A, Wash
B and DI Water) need to be replaced when the filters are
worn out. The software displays warning message boxes
for the filter in each reagent bottle when it detects a
problem or shows a trend of increased fill times during
fluid fill operations.
If an error is detected as described above, then a message
box titled “Filter Change Required” is displayed along
with the information on the specific dispense operation.
You should change all three filters when a warning is
displayed for any one of the three filters.
Refer to the section Replacing the Filter on page 248 in
Appendix F.
Chapter 9 | Troubleshooting 221
Wash/Scan Resume
If a run is aborted during fluidics processing, the instrument will place the aborted array plate into the
Scan Tray. To restart this process, remove the Axiom Array Plate from the Scan Tray and place it in its
protective blue base.
The step at which the run was aborted can be identified by:
Viewing the System Status window if you are aborting the last plate through the fluidics system.
Initiating the resume process.
1. System Setup tab: Select Wash/Scan Resume
2. Follow the prompts to unload and reload all drawers.
The trays will be loaded. It is up to you to determine whether or not to load fresh reagents or reuse the
trays already in the GeneTitan Multichannel Instrument. Base your decision upon the step where the
problem occurred.
To help ensure that the samples are processed correctly, we recommend that you:
1. Load new stain trays with fresh reagents.
2. Load a new Scan Tray.
We do not recommend the use of trays without reagents or holding buffers for steps that appear to have
already executed.
Resume Step
You must select the step at which you wish to resume plate processing. You can select any step that has
not yet been started.
For certain steps, you can enter a duration in seconds (even if the step requires > 1 hr to run, you must
enter the duration in seconds). You can set a step for less time than normal, but not for longer than the
normal duration.
Aborting a Run
Abort can take up to three minutes if a plate is in the Fluidics station. Status window Abort Requested
changes to Abort Completed.
Clamped Array-Plate -Hyb Tray sandwich aborted from the oven or from drawerIN are moved to
drawer 1.
Proceed as follows:
Use the Unload Plates option to remove the aborted plate(s).
Start another run which will force an unload of the aborted plate(s)
System-initiated
Power interruption
Plate loaded incorrectly
Equipment malfunction
The system will abort the processing. Follow the instructions displayed in the user interface.
User-initiated
Can abort processing of individual array plates.
If multiple plates are being processed, the gripper may continue to process the remaining array plates.

A
Fragmentation Quality Control Gel Protocol

Protocol for Running a Fragmentation Quality Control Gel
Equipment Required
Table A.1 Equipment required

Gel imager Your choice —
Pipette, single-channel P20 Your choice —
Plate centrifuge Your choice —
Vortex Your choice —
E-Gels and Reagents
Table A.2 E-Gel and reagents required

Mother E-Base Device EB-M03
Daughter E-Base Device Life Technologies EB-D03

(formerly Invitrogen)
E-Gel® 48 4% agarose gels G8008-04
TrackIt 25 bp DNA Ladder 10488-022
TrackIt Cyan/Orange Loading Buffer 10482-028
Consumables
Table A.3 Gel and reagents required

Adhesive film – use one of the following:

MicroAmp Clear Adhesive Film Applied Biosystems 4306311
Microseal 'B' Film MSB1001
Bio-Rad
Pipette Tips Same brand as pipette —

Diluting the TrackIt Cyan/Orange Loading Buffer

The following recipe is for preparing a 1000-fold dilution of the TrackIt Cyan-Orange Loading Buffer.
To dilute the TrackIt Cyan/Orange Loading Buffer:
1. Add 50 µL of TrackIt Cyan/Orange Loading Buffer to 49.95 mL nuclease-free water.
Total volume 50 mL.
2. Mix well.
3. Store at room temperature.
Fragmentation QC Gel Protocol

This protocol is based on running QC gels for 96 samples.
To run a fragmentation QC gel:
1. Tightly seal the gel QC plate prepared during automated or manual target preparation.
2. Vortex the center of the plate for 3 sec, and spin at 2000 rpm for 30 sec.
3. Power on two E-Bases.
4. Push the Power/Prg button on each to ensure the program is in EG mode (not EP mode).
5. Place the E-Gels onto the base units.
6. Remove two combs from each gel.
7. Load 20 µL from each well of the gel QC plate onto the gels.
8. Load 15 µL of diluted TrackIt 25 bp ladder into the marker wells (M).
9. Load 20 µL nuclease-free water into any unused wells.
10. Run the gels for 22 min.
11. Take a gel image.
Fragmentation QC gel images should look similar to the gel shown in Figure A.1.
Figure A.1 Example of a typical fragmentation QC E-gel
25 bp ladder 25 bp ladder
Fragments should fall between

125 bp and 25 bp.
125 bp
25 bp
125 bp
25 bp
B
Sample Quantitation after Resuspension

Protocol for Sample Quantitation after Resuspension
Equipment Required
The following equipment is required for this protocol.
Table B.1 Equipment required for sample quantitation after resuspension

Quantity Item
1 DTX 880 Multimode Detector with Genomic Filter Slide
Quantitate the Diluted Samples

During target prep, two plates of diluted samples are prepared: one for OD quantitation and one for a QC
gel to check the fragmentation reaction.
For OD quantitation, readings should be taken at 260, 280 and 320. See Suggested Protocol for OD
Quantitation Using the DTX 880 on page 227 for more information.
To quantitate the diluted samples prepared for OD quantitation:
1. Launch the Multimode Analysis Software.
2. When the Protocol Selection List is displayed, select the appropriate protocol.
3. Right click the protocol and select Run the selected protocol.
4. In the Result Name field, enter your experiment name.x
5. Click the Eject Plate Carrier icon.
6. Load the OD plate onto the DTX 880.
7. Click the Close Plate Carrier icon.
8. Click the Run the Selected Protocol icon at the bottom of the window.
When the protocol is finished running, a list of results is displayed. If you used the formula provided in
this appendix, two XML files are generated (Figure B.1). Open the ResultData file with Microsoft Excel
to view and assess the OD readings. RawData file information is included in the ResultData file.
Figure B.1
Assess the OD Readings

If using the formula provided in this appendix, the raw data is included in the final Result Data file.
Figure B.2 is an example of a Result Data file. Your OD readings should be similar to those displayed
below.
Figure B.2 Example of Result Data file with acceptable OD readings
OD Yield Assessment Guidelines

The measurement of the yield of DNA after resuspension of the pellets is an important QC checkpoint in
the Axiom assay. If the median yield for the plate is < 500 µg DNA per sample:
Pause the protocol.
Assess each of the steps performed to that point to determine the possible source of the low yields.
This DNA yield corresponds to an A260 value of 0.245 and an A260-A320 value of 0.210.
Appendix B | Sample Quantitation after Resuspension 227
Suggested Protocol for OD Quantitation Using the DTX 880

The formula suggested below requires six passes. The settings and formula are shown below.
Protocol Type — Analysis
Figure B.3 Protocol Type
General Settings — enter a name for the protocol
Figure B.4 General Settings

Technique Type — select Absorbance.
Figure B.5 Technique Type
Labware — x_Abs_Greiner 96 UV clear std (96 microplate format)
Figure B.6 Labware

Layout Settings — as appropriate for 96-array plates
Figure B.7 Layout Settings
Method Selection — add (+) the three formulas created on the Data Reduction Page to the Group 1 box.
Figure B.8 Method Selection

Data Reduction Page — create the formulas required for scans at 260, 280 and 320
This protocol consists of six passes. Click Add new Pass to create passes two through six, shown in these
figures below.
Figure B.9 Data Reduction Page — First Pass
Figure B.10 Data Reduction Page — Second Pass

Figure B.11 Data Reduction Page — Third Pass
Figure B.12 Data Reduction Page — Fourth Pass

Figure B.13 Data Reduction Page — Fifth Pass
Figure B.14 Data Reduction Page — Sixth Pass

Output Settings — Select Export to Microsoft® Excel and Show Result Viewer
Figure B.15 Output Settings
Save the protocol.
If Performing Sample Quantitation on a Plate Reader Other than the DTX880

Your plate reader should be calibrated to ensure accurate readings.
The total yield in µg per well can be calculated as:
(A - C)*D*V*E/P
Where:
A = the observed OD260
B = the observed OD320 (an estimate of a blank reading)
D = 120 (the net dilution factor when preparing the OD Sample plate as described in the Automated
or Manual protocol)
V = 115 (the volume of the sample in µL after the resuspension step)
E = 0.05 (the extinction coefficient of duplex DNA at 260 nm)
P = the optical path length for the plate type and plate reader used.
If your plate reader does not record the OD320, the OD260 of a blank solution of water only should
be used for the parameter "B" above.
The optical path length is dependent on the type of plate and spectrophotometer used. Check your
manufacturer's recommendations for the path length for your instrument and plate type or for
recommendations on how to measure this quantity. The SpectraMax Plus384, described as an
alternative spectrophotometer in the Axiom Site Prep Guide, P/N 702858, can employ an automated
path length detection system. Consult this instrument's manual for more information.
The resulting yield calculations can be compared against the typical yields shown in column H of
Figure B.2 on page 226 and against OD Yield Assessment Guidelines on page 226.

C
Rehybridization
Protocol for Rehybridizing Samples
Rehybridization
The target prep rehybridization protocol is used to help identify potential sources of problem in sample
failures.
You may occasionally encounter sub-optimal performance resulting in sample failures with the Axiom
Genotyping System. Failures that cannot be attributed to instrumentation issues may be due to either
sample quality and/or reagent and array issues.
To help isolate the potential cause of the problem, you may choose to perform a target prep re-
hybridization protocol. This is a protocol by which customers can re-process previously used
hybridization cocktails (that has been stored at -20 °C) in combination with a new array. The results of
this re-hybridization can indicate if the original sub-optimal performance was due to issues associated
with the array, array processing, the DNA target prep, or reagent quality.
Table C.1 shows conclusions that can be drawn from the results of the re-hybridization of Axiom
Genotyping AssayGenotyping Assay hybridization cocktail to the Axiom Genome-Wide Arrays.
Table C.1 Conclusions that can be drawn from the results of the re-hybridization of Axiom Genotyping Assay
hybridization cocktail to the Axiom Genome-Wide Arrays.
Results of Re-hybridization Details Conclusion
Pass/Significant improvement over Re-hybridization of hyb cocktail Problems in original hyb were not
original hybridization results in data that passes due to target prep. Possibly due to
performance metrics and is the array, array processing, and/or
significantly better than original reagents used for array
hybridization processing.
Fail/No improvement over original Re-hybridization of hyb cocktail Inconclusive. Target Prep may be
hybridization results in data that is not suspect however cannot exclude
significantly better than the problems with array and reagents,
original hybridization. or array processing issues.
Affymetrix has not established an upper limit for how long the hybridization plate can be stored at -20C
prior to re-hybridization. We have been successful in re-hybridizing plates that have been stored up to 11
days but individual results may vary as success depends on the quality of the original target prep and
amount of target available for re-hybridization.
Equipment and Consumables Required

Multi-channel pipette and tips, P100
PCR plate, BIO-RAD, P/N HSP9631
or
PCR Plate HSS-9601 stacked on a Costar serocluster round-bottom plate for the off-deck ABI 9700
and ABI 2720 thermal cycler when using the automated target prep methods
or
PCR Plate HSS- 9601 plate for use with manual target prep
Axiom
™ Genome-Wide or Custom myDesign™ Array Plate Kit (96 array)
Reagents
Modules 3 and 4 of the Axiom Reagent Kit
Water, nuclease-free ultrapure molecular-biology grade
Storing Hyb Trays for Rehybridization

To store a Hyb Tray:
1. When processing on the GeneTitan MC Instrument is complete, unload the Hyb Tray and tightly seal
it with an adhesive film.
2. Press the four corners and sides of the Hyb Tray to ensure that there is no open space between the seal
and the plate.
The plate must be well-sealed to prevent cross-contamination between samples.
3. Store the Hyb Tray at –20 °C.
Rehybridizing an Experiment
To rehybridize an experiment:
1. Remove the plate from the –20 °C freezer, and spin briefly to collect all of the liquid to the bottom
of the plate (bring up to 1000 rpm).
2. Slowly and carefully remove the seal to prevent cross-contamination.
3. Using a multichannel pipette, transfer the full volume from each well of the Hyb Tray to the
corresponding wells of a new PCR plate.
Volume per well should be ~45 µL.
4. To recover all of the remaining material from the Hyb Tray:
A. Add 50 µL of molecular biology grade water to each well using a multichannel pipette.
B. Pipet up and down 10 times to mix, rinsing each corner of the well as you mix.
C. Transfer the full volume from each well to the corresponding wells of the PCR plate.
5. Tightly seal the PCR plate with an adhesive film.
6. Vortex each corner of the plate at maximum speed.
7. Spin briefly again to collect all of the liquid to the bottom of wells.
8. Perform denaturation and transfer samples to a new Hybridization Plate:
If using the Biomek FXP Target Prep Express, run these methods:
Denature samples
Transfer denatured samples to hyb tray
If using the Manual Target Prep Workflow:

Perform Denaturation and Hybridization (see Stage 4 — Denaturation and Hybridization on
page 128)
IMPORTANT: Place a new Hyb Tray on the deck.
9. Transfer the Hyb Tray to the GeneTitan MC Instrument and:

A. Load the Hyb Tray and a new Array Plate onto the GeneTitan.
B. When hybridization is complete, prepare a new set of reagent plates using either:
The Biomek FXP Target Prep Express.
Manual Reagent Tray Prep (see Stage 5 — Manually Preparing Ligation, Staining, and
Stabilization Reagent Trays for the GeneTitan MC Instrument on page 134)
C. Transfer the reagent plates to the GeneTitan MC Instrument and finish processing the array plate.
D
Registering Samples in Affymetrix GeneChip® Command Console

Creating a GeneTitan Array Plate Registration File
A GeneTitan Array Plate Registration file is a Microsoft Excel spreadsheet that includes information on
the samples you are processing on a single array plate. This information includes the array plate format,
the array plate barcode, and sample file names so that you can track the samples that are loaded onto a
particular array plate.
The version of Microsoft Excel must be 1997-2000 (file extension is .xls; not .xlsx).
To create a GeneTitan Array Plate Registration file:

1. In AGCC Portal, open the Samples menu and select GeneTitan Array Plate Registration.
Figure D.1 Selecting Batch Register
2. Step 1 — Figure D.2 on page 238:

A. Select the array plate type.
B. Click Download.
Figure D.2 Selecting the type of array plate to be processed
3. Step 2 — complete the registration file as follows:

A. Click the Microsoft Excel box on the bottom bar of the monitor to open the Excel spreadsheet.
B. Enter a unique name for each sample (Sample File Name) and any additional information you
would like to include.
C. Do one of the following:
If you are ready to load the array plate onto the GeneTitan MC Instrument, scan the array plate
barcode and proceed to the next step.
If you are not ready to load the array plate onto the GeneTitan MC Instrument, proceed directly
to the next step.
Figure D.3 Entering sample information into a Batch Registration file.
4. Save the file as follows:

A. Open File > Save As.
B. Enter a name for the array plate registration file.
C. Click Save.
By default, the file is saved in the Affymetrix_Downloads folder.
5. Step 3 — when ready to load the array plate onto the GeneTitan MC Instrument:
A. Click the Browse button, navigate to the file, and click Open.
B. Scan the array plate barcode if not already scanned.
C. Click the Upload button (Figure D.4), wait for the information to load, then click the Save button
located at the bottom of the next page that is displayed.
Appendix D | Registering Samples in Affymetrix GeneChip® Command Console 239
If the samples are successfully registered, the message in Figure D.5 is displayed.
Figure D.4 Uploading the array plate registration file to AGCC.
Figure D.5 Array plate samples successfully registered.


E
Deionization Procedure for GeneTitan Trays and Covers

Use the following technique to destatic GeneTitan MC Stain Tray trays and lids.
IMPORTANT: Except for the HT array tray and the hybridization tray, you
must deionize all GeneTitan stain trays, stain tray covers and scan tray covers
using an anti-static gun. You must do this before you fill the trays with
reagents and before you place the covers on the trays. Deionization removes
the static electricity. The presence of static electricity on the underside of the
cover can cause the gripper to lift the tray along with the tray cover and can
result in an aborted run. See Figure E.1, Figure E.2 and Figure E.3.
Deionize the inner surface of each tray and lid:

The surface of the tray with the wells that will hold reagents.
The surface of the lid that will cover the reagents.
CAUTION: Do not deionize the scan tray or hybridization tray.
Figure E.1 Scan Tray with Cover. Deionize only the

cover.
Figure E.2 Stain Tray with Cover. Deionize the cover

and the tray.
Testing the Anti-Static Gun

Verify that the anti-static gun (PN 74-0014, Figure E.3.) is in working condition. You can use the
protective cap on the gun to determine if the anti-static gun is releasing ions. The procedure is as follows:
Keep the cap on the gun and press the trigger and release it. Observe the discharge through the viewing
slot on the cap of the anti-static gun. There is a visible light observed in the viewing window on the cap
when charged ions are discharged. If you cannot see the light, the gun may be un-usable and you should
replace it.
Each anti-static gun is capable of 50,000 trigger operations which is sufficient for approximately 200-250
runs on the GeneTitan instrument.
IMPORTANT: Make sure you remove the cap from the gun when you deionize a tray
or cover.
The following process provides guidance on how to use the anti-static gun on the stain and scan tray
covers only. See Figure E.3.
WARNING: The deionization steps 4 and 5 will damage the HT arrays on the plate. Before
using the anti-static gun, ensure that the HT array plates remain in their protective pouch and
placed away from the deionization area. You must place the scan tray and hybridization tray
away from the area where you are performing deionization.
1. Treat the plate or lid as if it were divided into 6 sections, and deionize as follows.
2. Place a Kimwipe on the benchtop.
3. Place the stain tray on a table top. Use the anti-static gun to aim at the center of each of the six sections
on a 96-well or 24-well cover or tray and pull the trigger. Ensure that a stream of ionized particles
settles on all wells of the stain tray or cover to dissipate the static electricity. Squeeze and release the
trigger slowly 3 times over each section (Squeeze for approximately two seconds and release for
approximately two seconds).
4. Place the stain tray cover with the flat surface facing upward on the Kimwipe.
Appendix E | Deionization Procedure for GeneTitan Trays and Covers 243
Aim the anti-static gun (P/N 74-0014) approximately one-half inch away from the flat surface and
pull the trigger. As you pull the trigger move the gun across the cover so that the stream of ionized
particles settles on all areas of the cover and dissipates the static electricity. Squeeze and release the
trigger slowly 3 times over each section (Squeeze for approximately two seconds and release for
approximately two seconds).
5. Place the treated cover or tray on the Kimwipe and lift it up.
If the Kimwipe does not adhere to the plastic, proceed with the step.
If the Kimwipe adheres to the plastic, then perform steps 3 and 4 again. If it continues to adhere to
the plastic, then the gun is not working and you should replace it.
Figure E.3 Removing the static charge from Stain Trays and lids.
Treat the inside surface of Stain Trays (right) and cover (left).
1 2
3 4
5 6
IMPORTANT: Remove static gun

cover before use.
If a Kimwipe sticks to treated surface,

treat again following this procedure.
If the Kimwipe still adheres, replace the
antistatic gun.

F
GeneTitan® MC Instrument Care

This chapter provides instructions on caring for and maintaining the instrument and on troubleshooting
if problems arise.
Always run a Shutdown protocol when the instrument will be off or unused overnight or longer. This
will prevent salt crystals from forming within the Fluidics system.
Always use deionized water to prevent contamination of the lines. Change buffers with freshly
prepared buffer at each system startup.
The GeneTitan® Instrument should be positioned on a sturdy level bench away from extremes in
temperature and away from moving air.
IMPORTANT: Before performing maintenance turn off power to the instrument to avoid
injury in case of an electrical malfunction.
Cleaning and Maintenance

The GeneTitan family of instruments require little in the way of customer maintenance. The instruments
must be kept clean and free of dust. Dust buildup can degrade performance. Wipe the exterior surfaces
clean using a mild dish detergent solution in water. Do not use ammonia based cleaners or organic
solvents such as alcohol or acetone to clean the system because they may damage the exterior surfaces.
The following tasks should be performed regularly to ensure the Imaging Device remains in working
order.
Monthly
Wipe down the outer surface of the Imaging Device with a dry cloth.
Every Six Months

Replace the cooling fan air filters at the rear of the instrument.
Replace the Micropore filters in the Wash A, Wash B, and Rinse bottles. If you run 4-8 plates/week then
the micro-pore filters need to be replaced more frequently.
Servicing the Outer Enclosure Fan Filters
Cleaning Schedule
The GeneTitan fan filter cartridge (Figure F.1) should be cleaned at least every 90 days of service. Note
that in some service locations, the presence of excessive dust or particulate matter may necessitate
cleaning the cartridge more often than 90 days.
A plugged filter cartridge can cause excessive temperatures within the machine that can cause unwanted
evaporation on test media.
Part details:
Affymetrix P/N: 01-0669
Number of parts per assembly: 3
Figure F.1 The GeneTitan filter cartridge
Cleaning Procedure
1. Slide the filter cartridge from the fan filter cartridge at the rear of the GeneTitan Instrument.
2. Submerse in clean DI water. Rinse and agitate gently to dislodge material.
3. Remove from water and dry with clean compressed air or towels.
4. When the filter cartridge is completely dry to the touch, re-install the cartridge.
Appendix F | GeneTitan® MC Instrument Care 247
Replacing the Bottle Filters

The bottles used in GeneTitan Instrument contain a filter to remove particulates that may exist in the
buffers and DI water. The filters in the GeneTitan fluidics bottles (Wash A, Wash B and DI Water) need
to be replaced when the filters are clogged.
The software displays warning message boxes for the filter in each reagent bottle when it detects a
problem or shows a trend of increased fill times during fluid fill operations.
If an error is detected as described above, then a message box titled “Filter Change Required” is displayed
(Figure F.2) along with the information on the specific dispense operation. You should change all three
filters when a warning is displayed for any one of the three filters.
Figure F.2 Filter change required messages
The message boxes displayed in Figure F.2 will provide information on fluid dispense errors that were
detected by the instrument for any of the bottles or when the instrument detects an increase in the amount
of time that is required to perform the fill operations.
NOTE: The reagent bottles are depressurized when this warning message is displayed. It is
safe to change the filters in all three fluidic bottles when this message is displayed.
After changing the filters in all three bottles using the procedure described below, please press the Yes
button to continue. If you choose to ignore the error message, press the No button. This warning message
will be displayed each time AGCC instrument control software is launched. You may also experience
data quality issues if particulate matter cannot be trapped by the filters if they are clogged.
We recommend that your site keep three spare filters in the event they need to be replaced. The procedure
for replacing the filters is simple.
Part details:
Affymetrix P/N: 01-0671
Figure F.3 Replacing the Filter
Buffer Supply Tube

Filter Holder
Filter
Removing and inspecting the Filter

1. Loosen and remove the cap on the bottle.
2. Carefully remove the filter from the end of the filter body.
3. Visually inspect the filter. If one of the filters appears to have a concentration of dirt or contaminate
in it, discard it and replace the filter with a new one.
Replacing the Filter

1. Insert the filter into the end of the filter body.
2. Replace the cap onto the bottle and tighten it.
3. Repeat for each bottle.
IMPORTANT: Replace one filter at a time to ensure the correct connection of the buffer
supply tube to its respective bottle. The color of the buffer supply tubing matches the bottle
color code.
Replacing the Xenon Lamp in the GeneTitan MC Instrument

This section applies to your site only if you have the GeneTitan Multi-Channel (MC) instrument. After
the normal life expectancy of the lamp has expired, the software application will alert you to the
requirement to replace the lamp. This procedure is simple but you must follow good health and safety
precautions.
Affymetrix Part Number: 01-0740
IMPORTANT: Please DO NOT try to replace the lamp when a plate is being processed either
in the fluidics or scanner system.
Lamp Life/Imaging Device Status Notices

The Imaging Status pane displays lamp life and Imaging Device status notices for the GeneTitan MC.
In normal operation, the pane displays the hours of life left in the lamp:
Figure F.4 Lamp Life above tolerance
It displays a red or yellow notice when the lamp life is getting short:
Figure F.5 Lamp Life above tolerance
It also displays a red notice when the Imaging Device is offline:
Figure F.6 Imaging Device Offline
NOTE: The 300 Watt Xenon lamp in the GeneTitan MC instrument is warranted for 500 hours.
The instructions to replace the lamp are available on the following page. After changing the
lamp, it is necessary to reset the lamp life clock manually.
WARNING: You must turn off the lamp using the power switch in the rear of the unit and
remove the power cord. Allow the lamp to cool before attempting to replace the lamp
Removing the Xenon Lamp

1. Unscrew the four retaining bolts. They should be finger tight (Figure F.7).
Figure F.7 Unscrewing the Bolts
Unscrew these four bolts.
2. Place each hand on each side of the blue plastic flange and lift out the lamp in a vertical motion
(Figure F.8). You must use both hands to remove the lamp successfully. Apply equal pressure on each
side of the lamp and gently lift.
Figure F.8 Lifting out the lamp
Replacing the Lamp
CAUTION: Ensure that you install the lamp in the correct orientation.
1. Hold the lamp by the blue plastic flanges. Ensure that the lamp bulb faces inward toward the reflecting
mirror (Figure F.9) and vertically insert the lamp (Figure F.10).
2. Replace the warning cover and hand tighten the bolts (Figure F.7).
Figure F.9 The Reflecting mirror
Reﬂecting
Mirror
Figure F.10 Inserting the Lamp
IMPORTANT: The lamp bulb faces

away from the fan and toward
the reﬂecting mirror.
Resetting the Lamp Counter

You must alert the software application that you have replaced the lamp so that the hours of the lamp
counter are reset to zero. This menu option is only available when the system is not processing any plates.
1. On the software application click Tools → Reset Counter for Life Remaining (Figure F.11).
Figure F.11 Inserting the Lamp
2. The software will display a message that allows you to change your mind.
Figure F.12 Are you Sure?
3. Click Yes if you want to reset the counter. The software will display a message that confirms that the
software has reset the counter (Figure F.13).
Figure F.13 The counter is reset.
Troubleshooting
This section provides instructions on how to identify and solve simple problems with the GeneTitan MC
Instrument. If a problem or error occurs that is not listed in this chapter contact a Affymetrix technical
support for assistance.
For software errors that do not involve hardware crashes the most common solution is to shut down the
application and then restart it. If the same error occurs shut down both the application and the computer
and then restart. If it still occurs shut down the GeneTitan MC Instrument and then restart.
Log Files
The log files are produced by different AGCC components. The logs provide a record of the tasks
performed by different components, such as the migration tools and installer. These log files provide
useful information for troubleshooting problems. These files may be requested by your field application
specialist (FAS), field service engineer (FSE), or the Affymetrix call center.
AGCC Log Files

The following files apply to the GeneTitan Instruments. All the AGCC log files from
C:\Command_Console\Logs The different log files include:
Systemlog.XML XML file with system information.
DEC.log Text file with information on the use of the Data Exchange Console.
DECError.log Text file with information on errors created while using DEC.
AGCC_LibFileImporter. log Text file with info on use of the Library File Importer.
(with date and time code)
Other AGCC Files

Your FAS and/or FSE may request you to send the following files for troubleshooting:
1. Library files (*.PARAMS, *.MASTER, *.WORKFLOW, *.SMD, *.MEDIA) located in
C:\Command_Console\Library, excluding the large analysis library files (CDF, PSI, GRC).
2. Provide a list of all sub folders and their contents under the library files folder located in
C:\Command_Console\Library. Please ensure there are no duplicate library files, as these can cause
problems.
3. AGCC system configuration file located at
C:\Command_Console\Configuration\Calvin.System.config
4. Pending job order files located in C:\Command_Console\Jobs
5. Other AGCC related information, such as:
A. The number of files under C:\Command_Console\Data, including sub directory.
B. If the system is a networked system or a standalone system.
C. Other applications installed on the system, such as antivirus application, MS Office, Internet
Explorer versions.
AGCC Log Files for GeneTitan MC Systems

Log files for the GeneTitan MC Instrument control processes are placed in subdirectories of the
Command Console\Logs\ folder. Affymetrix may need the following files for troubleshooting:
GeneTitan MC Fluidics
1. C:\Command_Console\Logs\96F\
A. subdirectories named by date (e.g., Log7-29-2009)
1) Collect all dated directories and contents since the GeneTitan application was started, not just
the date of the event (some logging goes into files from the date the application started so this
can be critical for us).
2) Absolutely required are all the log directories from the date the run was started to the date of
the event.
2. C:\Command_Console\Logs\96F\FluidicErrorLog - all files in this directory
GeneTitan MC Imaging Device

1. C:\Affymetrix\GeneChipHTScanControlMC\Log - collect all dated directories and contents since the
GeneTitan application was started
2. C:\Affymetrix\GeneChipHTScanControlMC\RunLog - collect all dated directories and contents
since the GeneTitan application was started
Problems and Solutions

This section provides instructions on how to identify and solve problems with the unit.
If problems arise with the instruments use the following tables to locate the description that matches the
problem. If you cannot find a solution call Affymetrix technical support for assistance.
For software errors that do not involve hardware crashes the most common solution is to shut down the
application and then restart it. If the same error occurs shut down both the application and the computer
and then restart. If it still occurs shut down the entire unit and then restart.
Insufficient Disk Space Notice

If there is not enough memory on the computer’s drives to save the data from an array plate, a notice
appears when:
You first initialize the software and instrument
You select arrays for imaging.
Figure F.14 Insufficient Disk Space Notice
If you see this notice, you will need to free up sufficient disk space before imaging starts.

Index
Numerics Cleaning 245
2 plate target prep and array processing 204 Cleaning and maintenance 245
3 array pates per week 209 Command Console 237
3 plate manual target prep 212 Consumables
Biomek 22
3 plate workflow
DNA amplification 41
oven temps 211
Fragmentation and purification 53
8 plate Workflow GeneTitan 28
GeneTitan MC Instrument 184 Manual target preparation 108, 116, 122, 129
8 plate workflow preparation for the GeneTitan MC Instrument 77
Biomek 183 rehybridization 235
Resuspension and hybridization preparation 62
A Covers 34
Aborting a process 156 Cyan/Orange Loading Buffer 224
Aborting a run 221
AGCC files 254
D
AGCC log files 254 Deionization 241
AGCC Log Files for GeneTitan MC Systems 255 Deionization procedure 242
Amplification master mix 113 Denaturation master mix 111
Amplification staging room 96 Denature samples 72
Amplified DNA DNA Amplification 40
thawing 185, 204, 212 summary 51
Anti-static gun Documentation 6
testing 242 Biomek 40
Array plate DTX 880 227
loading 163 Duration 11, 40, 53
Array processing Manual target preparation 107, 122, 128
GeneTitan MC Instrument 149 preparation for the GeneTitan MC Instrument 77
Automated target preparation
2 plates per week 203
2-plate target prep and array processing 204 E
8 plates per week 183 E-mail and telephone notifications 153
initial prep week 186
Equipment, Consumables and Reagents Required 12
Axiom assay 6
Axiom Ligate Buffer 80 F
Axiom Resusp Buffer 63
Failed messages 219
Axiom Wash A 80
Fan filters
Axiom™ Genotyping Assay 5 cleaning schedule 245
servicing 245
B Fluidic diagnostic messages 219
Batch registration file 14 Fragmentation
Biomek summary 59
8-plate workflow 183 Fragmentation and purification 53
troubleshooting 217 Fragmentation QC gel protocol 223
Bottle filters Fragmentation step 55
replacing 247
Frozen pellets 63
Fume hood 96
C
Care 245 G
GeneTitan MC Instrument 245
gDNA 13
Centrifuge and dry pellets 61
Gel protocol 223
Centrifuge and dry pellets and thaw reagents 124
GeneChip Command Console 237 GeneTitan MC Imaging Device 255

GeneTitan MC array processing
timing issues 211 M
GeneTitan MC Instrument Maintenance 245
8-plate workflow 184
Manual target prep
array processing 149
3 plate 212
cleaning and maintenance 245
continuing the workflow 181 Manual target Preparation
create and upload batch registration file 157 3 array pates per week 209
hybridization 158 Manual target preparation 95, 99
ligate, wash, stain and scan 172 control recommendations 96
load an array plate and hyb tray 163 denaturation and hybridization 128
load trays 174 DNA amplification 107
preparation 72 drying, resuspension and QC 122
proper installation of the GeneTitan tray consumables 173 duration 107
setup options 154 equipment and perform denaturation 130
setup the instrument 158 fragmentation and precipitation 115
shutting down 182 GeneTitan MC consumables 102
status window prompts and actions required 170 hybridization tray and load into GeneTitan MC 131
tray alignment and loading 149 ligation, staining, and stabilization reagent trays 134
troubleshooting 217, 254 master mixes and Axiom hold buffer 140
Xenon lamp 249 Plate requirements and recommendations 96
GeneTitan MC lamp 153 stain, ligation and stabilization master mixes 138
thermal cycler recommendations 97
Genomic DNA 9
timing issues 210
GWAS 5 Messages 218
Multiple plate workflows 6
H
Human genomic DNA 9 N
Hyb ready samples stored at -20 °C 130 Neutralization master mix 113
Hyb tray
loading 163
O
Hyb Trays
storing for rehybridization 181 OD quantitation 227
Hybridization OD readings 226
GeneTitan MC Instrument 158 OD yield assessment guidelines 226
Hyb-Wash 155 Oven temperatures for 3 plate workflow 211
I P
Insufficient disk space notice 255 Pate centrifuge 17
Pipette Tip Usage 17
L Pipettes and pipetting 100
Labeling Plate reader other than the DTX880 233
GeneTitan hybridization and reagent trays 152 Precautions 7
Labeling GeneTitan hybridization and reagent trays 106 Precipitation 61
Lambda LS Xenon Arc Lamp Prepare GeneTitan reagent plates 72
replacing the lamp 249 Pre-Run Checklist
Lamp DNA amplification 42
Lambda LS Xenon Arc Lamp 249 Fragmentation and purification 54
Lamp counter preparation for the GeneTitan MC Instrument 78
resetting 253 Resuspension and hybridization preparation 63
Lamp life/Imaging Device status notices 249 Problems and solutions 255
Ligate, Wash, Stain and Scan 172 Procedure
Light curtain 16 deionization 241
Load an array plate and hyb tray 163 procedures 99
Load trays on to GeneTitan MC Instrument 174 Protocol
rehybridization 235
Loading a second array plate and hyb tray 168
Log files 254
GeneTitan MC Fluidics 255
Index 259
Q U
QC control gel protocol 223 Unload Plates 155
Quantitation 225
Quantitation and fragmentation QC checks 127 W
Wash/Scan Resume 221
R Wash-Scan 155
Reagent block template 40 Wash-Scan-Resume 155
References 6 Workflow 181
Registering samples 237
Registration file 237
Rehybridization 235
storing hyb trays 236
Rehybridizing an experiment 236
Related documentation 6
Resuspension and hybridization master mix preparation 126
Resuspension and Hybridization Preparation
summary 69
S
Safety warnings 7
Safety warnings and precautions
Manual target preparation 96
Sample plate stored at –20 °C
preparation 81
Sample quantitation 225
Scan 155
Seal, vortex and spin 99
Setup options
GeneTitan MC Instrument
154
Shutting down the GeneTitan MC Instrument 182
Simultaneous 8-plate workflow 192
Software default settings 17
Stain trays and covers 151
Storing Hyb Trays for rehybridization 181
T
Target preparation with Biomek 15
Thawing frozen plates of amplified DNA 185
Timing issues
Manual target Prep 210
Transfer denatured samples to Hyb Tray 72
Trays 125
alignment and loading 32
alignment and loading into GeneTitan MC Instrument 149
labeling 35
labeling hyb trays 35
labeling stain trays 36
loading in GeneTitan MC Instrument 36
stain trays and covers 34
Troubleshooting 217

Axiom Genotyping Assay User Manual

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User Manual

Axiom™ Genotyping Assay

P/N 702830 Rev 4

Chapter 1 About the Axiom™ Genotyping Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Chapter 2 Genomic DNA Preparation and Requirements . . . . . . . . . . . . . . . . . . . . . . . 9

Chapter 3 Axiom™ Genotyping Assay:

Chapter 4 Axiom Genotyping Assay:

Chapter 5 Axiom™ Genotyping Assay:

Chapter 9 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

Appendix A Fragmentation Quality Control Gel Protocol. . . . . . . . . . . . . . . . . . . . . . . 223

Appendix B Sample Quantitation after Resuspension. . . . . . . . . . . . . . . . . . . . . . . . . . 225

Appendix C Rehybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Appendix D Registering Samples in Affymetrix GeneChip® Command Console . . . . . 237

Appendix E Deionization Procedure for GeneTitan Trays and Covers . . . . . . . . . . . . . 241

Appendix F GeneTitan® MC Instrument Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

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About the Axiom™ Genotyping Assay

Overview of the Axiom Assay

FXP Target Prep Instrument on page 15)

Running Multiple Plate Workflows

 Hybridization in GeneTitan is 23.5 hours.

Safety Warnings and Precautions

CAUTION: All chemicals should be considered as potentially hazardous. We, therefore,

WARNING: The following components contain harmful or toxic ingredients:

 Axiom HybSoln 2: 100% Formamide

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Genomic DNA Preparation and Requirements

Sources of Human Genomic DNA

Assessing the Quality of Genomic DNA Using 1% Agarose E-gels

Equipment and Reagents Recommended

Table 2.1 E-Gel® and reagents required

Mother E-Base Device EB-M03

Daughter E-Base Device Life Technologies EB-D03

E-Gel® 96 High Range DNA Marker 12352-019

Guidelines for Preparing the Genomic DNA Plate

is 5 µL, add 3 µL of RediLoad, and bring to 20 µL total by adding 12 µL of H2O).

To Run the E-Gel:

Genomic DNA Extraction/Purification Methods

Genomic DNA Cleanup

Genomic DNA Preparation

Equipment, Consumables and Reagents Required

Equipment and Consumables

As required Adhesive seals for plates

1 Ice bucket, filled with ice

 Optional: multi-channel P10 or P20

As required Pipette tips

1 Plate, deep well:

Table 2.3 Reagents required for Genomic DNA Preparation.

From the Axiom Reagent Kit

 Axiom Reference Genomic DNA 103 (use as a positive control) — —

1. Thaw Samples and Control

2. Quantitate and Dilute gDNA

4. Seal, vortex and spin.

3. Aliquot the Diluted Samples and the Control

FXP Target Prep Instrument on page 15)

5. Create a Batch Registration File

Figure 2.2 Example of a batch registration file

 Breaking the Light Curtain on page 16

 Plate Centrifuge on page 17

 Labeling GeneTitan Hybridization and Reagent Trays on page 35

 Pipette Tip Usage on page 17

 Set the Biomek Software Default Settings on page 17

 Equipment, Consumables, Labware and Reagents Required on page 22

 Reagent Block Template on page 40

Hybridization in GeneTitan is 23.5 hours.

Axiom HybSoln 2: 100% Formamide

Optional: multi-channel P10 or P20

Axiom Reference Genomic DNA 103 (use as a positive control) — —

Breaking the Light Curtain on page 16

Plate Centrifuge on page 17

Labeling GeneTitan Hybridization and Reagent Trays on page 35

Pipette Tip Usage on page 17

Set the Biomek Software Default Settings on page 17

Equipment, Consumables, Labware and Reagents Required on page 22

Reagent Block Template on page 40

Related Biomek FXP Target Prep Express Documentation on page 40

Stage 1 — DNA Amplification on page 40

Transfer denatured samples to Hyb Tray

Prepare GeneTitan reagent plates

Custom run options

Deck configuration options

ABI 9700 with a gold, silver or aluminum block

Quarter module divided by width,

Black Scan PN 202096 The Black Scan tray

Scan Tray Scan Tray 501006 The GeneTitan Scan tray

GeneTitan 202757 The GeneTitan scan and