TM

GeneiPure Plasmid Purification GeNeiTM TM

GeneiPure Plasmid Purification GeNeiTM

TM
GeneiPure Plasmid
Purification Kit
Manual

2115200021730
Cat No. 2115200031730
2115200051730
Revision No.: 00050516

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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

CONTENTS

Page No.

v Introduction 1

v Description 1

v Material Supplied 3

v Protocol 5

v Trouble Shooting 11

v Ordering Information 14

v Other Related Products 14

v Flow Chart 15

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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

Introduction:
A plasmid is a small, Circular, double-stranded DNA molecule
that is distinct from a cell’s chromosomal DNA. A plasmid is
an independent, self-replicating DNA molecule that carries
only a few genes. Plasmids naturally exist in bacterial cells,
and they also occur in some eukaryotes. Plasmids are easy
to manipulate and isolate (Alkali lysis method or Boiling
method).
Scientists have taken advantage of plasmids to use them as
tools to clone, transfer, and manipulate genes. Plasmids that
are used experimentally for these purposes are called vectors.
A foreign DNA fragment can be inserted into a plasmid vector,
creating a recombinant plasmid. This plasmid can be
introduced into a bacterium by the process of transformation.
Since bacteria divide rapidly, they serve as factories to copy
DNA fragments in large quantities. Vectors have selectable
antibiotic marker, an origin of replication and a multiple cloning
site (i.e site for various restriction endonucleases). Expression
vectors contain promoter sequence that drives the expression
of transgene. Besides this plamid vectors may contian genetic
markers, epitope, protein purification tags and reporter genes.
Thus plasmids are importaint genetic engineering tools as
they are used multiply, manipulate, study and express genes.

Description:
TM
GeneiPure Plasmid Purification Kit is designed for rapid
purification of high quality plasmid DNA from bacterial
cultures. Bacteria are lysed by the Lysis Buffer and the
addition of high salt containing buffer facilitates neutralization
TM
and binding of DNA to the GeneiPure Column. Contaminants
like metabolites, proteins, salts and other small molecules
are removed by the two-step wash procedure with Wash
Buffer I and Wash Buffer II. Pure Plasmid DNA is eluted using
Elution Buffer under low ionic and slightly alkaline conditions.
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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

Highlights: Materials Supplied:

• Purify high copy and low copy plasmids The list below provides information about the materials
• Plasmids upto 15kb can be isolated supplied in the kit. The components should be stored as
• No detectable genomic DNA or RNA contamination suggested.
• Upto 80% recovery of DNA compared to conventional miniprep
• High recovery: High copy - 10-15 µg of DNA from 1-5 ml Quantity
bacterial culture .
Materials 2115200021730 2115200031730 2115200051730 Store
Low copy - 5-10 µg DNA from 5-10 ml
(20 preps.) (50 preps.) (250 preps.)
bacterial culture.
• Fast and simple- 12 preps/25 minutes. RNase A (lyophilized) 0.5 mg 1.25 mg 1.25 x 5 mg -20°C
• Culture volumes upto 10 ml can be processed owing to the high
TM Solution G1 5 ml 12.5 ml 65 ml 4°C
binding capacity of the new GeneiPure Column
Wash Buffer II 3 ml 7.5 ml 40 ml 4°C
Applications: (Concentrate)
Purified plasmid prepared is suitable for all downstream applications Elution Buffer 4 ml 10 ml 50 ml 4°C
like restriction digestion, transformation, PCR, sequencing etc.
Solution G2 5 ml 12.5 ml 65 ml RT

Time Required: 12 preps/25 minutes Solution G3 7 ml 17.5 ml 90 ml RT

Wash Buffer I 10 ml 25 ml 125 ml RT

M 1 2 3 4 5 6 M
GeneiPure™ DNA 20 Nos 50 Nos 250 Nos. RT
Spin Columns
Marker (M): StepUp™ 1 kb DNA Ladder
Lane 1: 2 kb Plasmid DNA (Low Copy) Collection Tubes 20 Nos 50 Nos 250 Nos. RT
Lane 2: 3 kb Plasmid DNA (High Copy)
Note:
Lane 3: 4 kb Plasmid DNA (High Copy) • Room Temperature not to exceed 20-25°C.
Lane 4: 5 kb Plasmid DNA (High Copy)
• The Kit provides sufficient components to purify high copy
Lane 5: 6 kb Plasmid DNA (High Copy) plasmid from 50 samples. For Low copy protocol refer to
Lane 6: 10 kb Plasmid DNA (Low Copy) Page 8.

Materials Required but not provided:

Fig: Analysis of various sizes of Plasmid DNA
purified using GeneiPure™ Plasmid Purification Kit Equipment : Microcentrifuge, Vortex, Dry Bath/Water Bath
Reagent : Absolute ethanol (96-100%), Lysozyme,
Glass Distilled Water (Sterile)
Other Requirements : 1.5 ml vials (Sterile)
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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

Notes: Protocol:
• Read the entire procedure carefully prior to starting For High Copy Number Plasmids
the experiment For Gram Negative Bacteria:
• Before using the kit add 1 ml of Solution G1 to the 1. Pick a single bacterial colony from a freshly streaked
RNase A vial and mix thoroughly. Transfer this to the plate and inoculate in 1-5 ml of LB medium. Incubate at
Solution G1 bottle and mix well. Record the date of 37°C for 16 hours with rigorous shaking.
addition of RNase A on the label and store at 4°C. (Use Note:
within 3 months). • Do not grow for more than 16 hours as this may
• Do not freeze Solution G1 after addition of RNase A. reduce the plasmid yield.
• Wash Buffer II is supplied as concentrate. Dilute only 2. Centrifuge 1-5 ml of culture at 6000-8000 rpm to pellet
required amount with 4 volumes of absolute ethanol just the bacterial cells.
before use. Do not store diluted Wash Buffer II. 3. Remove the culture supernatant carefully.
• Solution G2 may form a precipitate if stored at lower 4. Resuspend the pellet in 250 µl Solution G1 with RNaseA
temperature (below 15°C). Incubate the bottle at 37°C and mix well by vortexing.
till the precipitate dissolves. Mix well to get a uniform Note:
solution, before use. • No lumps should be observed before proceeding
• All centrifugation steps are to be carried out in a to the next step.
conventional microcentrifuge at room temperature. • For 10 ml culture use 500 µl of Solution G1.
5. Add 250 µl of Solution G2. Mix gently by inverting the
tube 6-8 times for 5 min at room temperature.
Note :
• Do not vortex. Do not allow the reaction to
proceed for more than 5 minutes at room
temperature.
• For 10ml culture use 500 µl of Solution G2.
6. Add 350 µl of Solution G3 and mix by inverting the tubes
(6-8 times). Centrifuge at 11,000 rpm for 10 minutes at
room temperature.
Note:
• Do not vortex. Centrifugation time may vary from
5-10 minutes depending on the culture volume.
• For 10 ml culture use 500 µl of Solution G3.

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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM
TM
7. Collect the supernatant carefully in a fresh 1.5 ml vial. In 15. For Elution place the GeneiPure DNA Spin Column in a
case the supernatant is not clear, repeat centrifugation fresh 1.5 ml vial and add Elution Buffer to the center of
TM
for additional 5 minutes. the GeneiPure Spin Column.
Note: Debris in the supernatant may clog the • For High Yield: Perform first step of elution with
column. 50 µl Elution Buffer. Incubate at room temperature for
8. Place GeneiPure™ DNA Spin Column in the 2 ml 1 minute and centrifuge at 11,000 rpm. Repeat the
Collection Tube. Load the supernatant, 400 µl at a time. elution step with additional 50 µl of Elution Buffer
Centrifuge at 11,000 rpm for 1 minute. Discard the flow • For High Concentration: Perform first step elution
through, before proceeding to the next centrifugation step. with 30 µl Elution Buffer. Incubate at room temperature
9. Wash with 500 µl of Wash Buffer I and centrifuge at 11,000 for 1 minute and centrifuge at 11,000 rpm. Repeat
rpm for 1 minute. Discard the flow through and place the elution step with additional 50 µl of Elution Buffer.
GeneiPure™ DNA Spin Column back in the same 16. Store the eluted DNA at -20°C.
Collection Tube.
10. Dilute one volume of Wash Buffer II (concentrate) with For Gram Positive Bacteria:
4 volumes of absolute ethanol e.g. for 6 preps dilute 1. Follow step 1-3 of protocol for Gram Negative Bacteria.
0.6 ml of Wash Buffer II with 2.4 ml of absolute ethanol, 2. Add lysozyme at a final concentration of 2 mg/ml to
mix thoroughly. appropriate volume of Solution G1.
11. Wash the GeneiPure™ DNA Spin Column with 500 µl of 3. Proceed with step 4 of protocol for Gram Negative
Wash Buffer II. Bacteria.
12. Centrifuge for 2 minutes at 11,000 rpm to remove traces
of Wash Buffer II. Discard the Collection Tube.
Note: Ensure that the base of the column does not
come in contact with the flow through while
removing it from the centrifuge and the Collection
Tube.
13. Open the GeneiPure™ DNA Spin Column and place in a
fresh 1.5 ml vial. Incubate for 2 minutes at 70°C to ensure
complete removal of ethanol.
14. Take required amount of Elution Buffer in a 1.5 ml vial
(sterile) and warm at 70°C in a dry bath for 5 minutes.

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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

Protocol: 8. Place GeneiPure™ DNA Spin Column in the 2 ml

For Low Copy Number Plasmids Collection Tube. Load the supernatant, 400 µl at a time.
Centrifuge at 11,000 rpm for 1 minute. Discard the flow
For Gram Negative Bacteria: through, before proceeding to the next step.
1. Pick a single bacterial colony from a freshly streaked 9. Wash with 500 µl of Wash Buffer I and centrifuge at
plate and inoculate into 5-10 ml of LB medium. Incubate 11,000 rpm for 1 minute. Discard the flow through and
at 37°C for 16 hours with rigorous shaking. place the GeneiPure™ DNA Spin Column back in the
Note: Do not grow for more than 16 hours as this same Collection Tube.
may reduce the plasmid yield. 10. Dilute one volume of Wash Buffer II (concentrate) with
2. Centrifuge 5-10 ml of culture at 6000-8000 rpm to pellet 4 volumes of absolute ethanol e.g. for 6 preps dilute
the bacterial cells. 0.6 ml of Wash Buffer II with 2.4 ml of absolute ethanol.
3. Remove the culture supernatant carefully. Mix thoroughly.
4. Resuspend the pellet in 500 µl Solution G1 with 11. Wash the GeneiPure™ DNA Spin Column with 500 µl of
RNase A and mix well by vortexing. Wash Buffer II.
Note: No lumps should be observed before 12. Give a final spin for 2 minutes at 11,000 rpm to remove
proceeding to the next step. traces of Wash Buffer II. Discard the Collection Tube.
5. Add 500 µl of Solution G2. Mix gently by inverting the Note: Ensure that the base of the column does not
tube 6-8 times. come in contact with the flow through while
removing it from the centrifuge and the Collection
Note: Do not vortex. Do not allow the reaction to Tube.
proceed for more than 5 minutes at room
temperature. 13. Open the GeneiPure™ DNA Spin Column and place it in
a fresh 1.5 ml vial. Incubate for 2 minutes at 70°C to
6. Add 500 µl of Solution G3 and mix by inverting the tube ensure complete removal of ethanol.
(6-8 times). Spin at 11,000 rpm for 10 minutes at room
temperature. 14. Take required amount of Elution Buffer in a 1.5 ml vial
(sterile) and warm at 70°C in a dry bath for 5 minutes.
Note: Do not vortex. Centrifugation time may vary
from 5-10 minutes depending on the culture volume.
7. Collect the supernatant carefully in a fresh 1.5 ml vial. In
case supernatant is not clear, repeat centrifugation for
additional 5 minutes.
Note: Debris in the supernatant may clog the
column.

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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

15. For Elution place the GeneiPure™ DNA Spin Column in Trouble Shooting:
a fresh 1.5 ml vial and add Elution Buffer at the center of Observation: Low or no plasmid yield (Band intensity is
the GeneiPure™ DNA Spin Column. low)
• For High Yield: Perform first step of elution with
50 µl Elution Buffer. Incubate at room temperature for Possible Cause Suggestions
1 minute and centrifuge at 11,000 rpm. Repeat elution Inappropriate dilution Dilute 1 volume of Wash Buffer II with
step with additional 50 µl of Elution Buffer. of Wash buffer 4 volumes of absolute ethanol, just
• For High Concentration: Perform first step of elution before use.
with 30 µl Elution Buffer. Incubate at room temperature Insufficient drying of Centrifuge for 5 minutes at 11,000
for 1 minute and centrifuge at 11,000 rpm. Repeat GeneiPure™ DNA rpm or incubate column for
elution step with additional 50 µl of Elution Buffer. Spin Column 2-5 minutes at 70°C before elution
16. Store eluted DNA at -20°C. to remove Wash Buffer completely
For Gram Positive Bacteria: Add Elution Buffer at the center of
TM
Improper elution the GeneiPure DNA Spin Column
1. Follow step 1-3 of protocol for Gram Negative Bacteria. and incubate at room temperature
2. Add lysozyme at final concentration of 2 mg/ml to for 1 minute before centrifugation.
appropriate volume of Solution G1.
Do not incubate culture for more
3. Proceed with step 4 of protocol for Gram Negative Overgrown bacterial than 16 hours at 37°C.
Bacteria. culture
When using a rich medium like TB
(Terrific Broth), grow the culture for
less than 12 hours.

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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

Observation: Incomplete lysis of bacterial cells Observation: Sequencing reaction not working
Possible Cause Suggestions Possible Cause Suggestions
Precipitation of Solution G2 forms a precipitate If Elution Buffer other Repurify the DNA using a new
Solution G2 when stored at low temperature. than the one GeneiPure™ DNA Spin Column and
Incubate at 30-40°C for 5 minutes provided in the kit is elute with Elution Buffer provided in
and mix well before use. used, EDTA may the kit.
Very high cell Do not overgrow the culture. Cell inhibit sequencing
Precipitate with ethanol and
density density may be too high when using reaction
resuspend in the Elution Buffer
rich medium like TB. provided in the kit.
Cells not Ensure the cell pellet is completely
resuspended Quality and Check the concentration and quality
resuspended before adding Solution
properly concentration of of DNA on agarose gel before
G2. No lumps should be visible
DNA proceeding for sequencing reaction.

Ethanol in eluted Centrifuge for 2 minutes after

Observation: Poor Plasmid quality (DNA seen as a smear DNA washing with Wash Buffer II to
on the gel). remove residual ethanol.
Possible Cause Suggestions Additionally, incubate at 70°C for
Nicked plasmid DNA Do not incubate in Solution G2 for 2-5 minutes before elution
more than 5 minutes
Genomic DNA Chaotropic salt in Dilute one volume of concentrated
Genomic DNA is sheared if vortexed
contamination eluted DNA Wash Buffer II with 4 volumes of
or mixed vigorously after addition of
absolute ethanol before use.
Solution G2. Mix gently.

Improper storage of Quantitate the plasmid on agarose

purified plasmid gel and store at -20°C for long term
storage

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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

Ordering Information Flow Chart

For High copy Number Plasmid: Alkali Lysis:
Product Size Cat # To 1-5 ml E.coli Bacterial pellet add 250 µl
G1. Mix by inverting. Add 250 µl G2 mix by
GeneiPureTM 1 EA 2115200021730 inverting. Add 350 µl G3. Mix by inverting.
Plasmid Purification Kit For Low copy Number Plasmid: 5-10 ml
(20 Preparations) Bacterial pellet + 500 µl G1 + 500 µl G2 + 500
TM
µl G3. Spin at 11,000 rpm 5-10 mins at RT.
GeneiPure 1 EA 2115200031730
Plasmid Purification Kit
(50 Preparations) Clarification of Lysate
TM
GeneiPure 1 EA 2115200051730
Plasmid Purification Kit
(250 Preparations) Load: Complete sample (400 µl at a time),
spin at 11,000 rpm for 1 min. Discard flow
through.

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2115700031730 GeneiPure Plant Genomic DNA Purification Kit Wash II: Add 500 µl diluted Wash Buffer II,
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GeneiPure Blood Genomic DNA Purification Kit spin at 11,000 rpm for 2 min. Discard
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2115300031730 GeneiPure Quick PCR Purification Kit
2150380501730 RNase A
2150100011730 Proteinase K
Incubate at 70°C for 2 min. Elute: 50 µl pre-
warmed Elution Buffer. Spin at 11,000 rpm
Email: for 1 min. Repeat for high yield.
Sales: sales@geneilabs.com
Restriction Digestion
Customer Support: techsupport@geneilabs.com Sequencing Transformation
Cloning PCR

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GeneiPure Plasmid Purification GeNeiTM TM
GeneiPure Plasmid Purification GeNeiTM

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