QUICK REFERENCE

TaqPath™ ProAmp™ Master Mixes

Genotyping and copy number variation PCR workflows
Pub. No. 100040972 Rev. D
Note: For safety and biohazard guidelines, see the “Safety” Set up the PCR reactions
™ ™
appendix in the TaqPath ProAmp Master Mixes User Guide
1. Prepare the required number of reactions according to the
(Pub. No. MAN0015758). Read the Safety Data Sheets (SDSs) and
appropriate table, plus 10% overage.
follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Table 1 PCR reactions for genotyping experiments
This Quick Reference is intended as a benchtop reference Volume per reaction
™ ™
for experienced users of TaqPath ProAmp Master Mix and Component
™ ™ 5 µL 10 µL 25 µL
TaqPath ProAmp Multiplex Master Mix. For detailed instructions,
™ TaqPath™ ProAmp™ Master
supplemental procedures, and troubleshooting, see the TaqPath 2.5 µL 5.0 µL 12.5 µL
™ Mix
ProAmp Master Mixes User Guide (Pub. No. MAN0015758).
TaqMan™ SNP Genotyping
™ ™
TaqPath ProAmp Master Mix is formulated with ROX dye
™ 0.25 µL 0.5 µL 1.25 µL
™ ™
Assay[1] (20X)
(absorption 575 nm, emission 602 nm). TaqPath ProAmp Multiplex up to up to
™
Master Mix is formulated with MUSTANG PURPLE dye (absorption Genomic DNA -or- NTC up to 4.5 µL
2.25 µL 11.25 µL
647 nm, emission 654 nm). to 10 µL to 25 µL
Nuclease-Free Water to 5 µL total
Both formulations contain heat-labile uracil-DNA glycosylase (UNG) total total
and dUTP. Heat-labile UNG degrades any carryover PCR products, Total volume 5 µL 10 µL 25 µL
helping to prevent downstream contamination and possible false [1] For Research Use Only. Not for use in diagnostic procedures.
positives. The heat-labile enzyme is active at room temperature and is
completely inactivated during the initial ramp to the 95°C hold step. Table 2 PCR reactions for copy number experiments
Volume per reaction
Procedural guidelines Component
10 µL 20 µL
Guidelines for DNA template input TaqPath™ ProAmp™ Master
5.0 µL 10.0 µL
Mix
• All wells that use the same assay must contain similar amounts of
sample DNA. TaqMan™ Copy Number
0.5 µL 1.0 µL
Assay[1] (20X)
• For genotyping experiments, use a final DNA concentration of at
TaqMan™ Copy Number
least 0.2 ng/µL for each reaction. 0.5 µL 1.0 µL
Reference Assay[1] (20X)
• For copy number experiments, use a final purified DNA
Genomic DNA -or- NTC up to 4 µL up to 8 µL
concentration of 1 ng/µL for each reaction.
Nuclease-Free Water to 10 µL total to 20 µL total
Guidelines for PCR reactions Total volume 10 µL 20 µL
• For copy number experiments, we recommend four replicates of [1] For Research Use Only. Not for use in diagnostic procedures.
each reaction. 2. Mix the components thoroughly, then centrifuge briefly to spin
• Reactions can be prepared depending upon experimental down the contents and eliminate air bubbles.
requirements. Scale the components according to the number of 3. Add the appropriate volume of each reaction to each well of an
reactions and include 10% overage. optical plate.
• For reaction volumes that are different from those detailed, scale 4. Seal the plate with an optical adhesive cover, then centrifuge
all components proportionally and include 10% overage. briefly to spin down the contents and eliminate air bubbles.
• For genotyping experiments, no template control (NTC) reactions Store the reaction plate for up to 72 hours at room temperature.
are required to call the genotype. NTC reactions can also be used
to identify PCR contamination.
NTC reactions contain all reaction components (master mix,
assays, water) except DNA sample.

For Laboratory Use.

Set up and run the real-time PCR instrument Perform allelic discrimination
1. Load the reaction plate in the real-time PCR instrument. For genotyping experiments, perform allelic discrimination using a
2. Set the appropriate experiment settings and PCR thermal cycling post-read temperature of 60°C.
conditions. See the instrument user guide for further information.
Note: Heat-labile UNG is active during the reaction setup and
is completely inactivated during the first ramp to the 95°C hold Analyze the results
step. Data analysis varies depending on the instrument.
™ ™
Table 3 Genotyping experiments: standard cycling See the TaqPath ProAmp Master Mixes User Guide
(Pub. No. MAN0015758) and the instrument user guide.
Step Temperature Time Cycles
Pre-Read 60°C 30 seconds Ordering information
Initial denature / Hold
95°C 5 minutes Table 6 TaqPath™ ProAmp™ Master Mix formulations
Enzyme activation
Denature 95°C 15 seconds TaqPath™ ProAmp™ Master TaqPath™ ProAmp™
40 Mix Multiplex Master Mix Amount
Anneal / Extend 60°C 60 seconds[1]
with ROX™ with MUSTANG PURPLE™
Post-Read 60°C 30 seconds Hold
[1]
A30865 A30868 1 × 1 mL
For Drug Metabolizing Enzyme (DME) assays, set duration to 90 seconds.
A30866 A30869 1 × 10 mL
Table 4 Genotyping experiments: fast cycling
A30871 A30873 2 × 10 mL
Step Temperature Time[1] Cycles A30867 A30870 1 × 50 mL
Pre-Read 60°C 30 seconds A30872 A30874 2 × 50 mL
Initial denature / Hold
95°C 5 minutes
Enzyme activation
Denature 95°C 5 seconds
40
Anneal / Extend 60°C 30 seconds
Post-Read 60°C 30 seconds Hold
[1] Optional cycling for Fast 96-well or 384-well plates only.
Table 5 Copy number experiments: standard cycling
Step Temperature Time Cycles
Initial denature /
95°C 10 minutes Hold
Enzyme activation
Denature 95°C 15 seconds
40
Anneal / Extend 60°C 60 seconds
3. Set the reaction volume appropriate for the reaction plate.
4. Start the run.

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The information in this guide is subject to change without notice.

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Revision history: Pub. No. 100040972
Revision Date Description
D 20 January 2022 Change manufacturer to Austin, TX.
C 04 September 2018 Change manufacturer to Vilnius
Change multiplex master mix name; add final DNA concentration guidelines; add UNG and DME assays information; add pre- and
B 11 July 2016
post-read steps to genotyping thermal cycling conditions
A April 2016 New document.

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20 January 2022