TM

GeneiPure Bacterial DNA Purification Kit GeNeiTM TM

GeneiPure Bacterial DNA Purification Kit GeNeiTM

TM
GeneiPure Bacterial DNA
Purification Kit
Manual

Cat No. 2115900031730

Revision No.: 00050516
 Genei Laboratories Private Limited, 2016  Genei Laboratories Private Limited, 2016
 TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM

CONTENTS

Page No.

v Description 3

v Material Supplied 5

v Protocol 7

v Trouble Shooting 10

v Ordering Information 12

v Other Related Products 12

v Flow Chart 13

 Genei Laboratories Private Limited, 2016  Genei Laboratories Private Limited, 2016 1
 TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM

Description:
TM
GeneiPure Bacterial DNA Preparation Kit provides a
rapid, simple method for the isolation of ready to use genomic
DNA from gram positive & gram negative bacteria.

Bacterial samples are lysed using Proteinase K and Lysis

TM
Buffers and then loaded on to GeneiPure Column. DNA
selectively binds to the membrane under high salt conditions
while the contaminants pass through. Other impurities are
completely removed by an effective wash step and pure nucleic
acid is eluted in minimal volume of Elution Buffer.

The purified genomic DNA is ready-to-use in down stream

applications like:
• PCR
• Cloning
• Restriction digestion
( Refer Fig 1 & 2)

Highlights:
• Improved yield, upto 8-10 µg of DNA from 0.5-1 ml of E.coli
culture and upto 5-10 µg of DNA from 1-1.5 ml Bacillus
subtilis
• Unique lysis buffer optimized for Bacterial DNA Purification

Time Required:
Hands on time: 1 hour
Total time: 2.30-3 hours

 Genei Laboratories Private Limited, 2016 2  Genei Laboratories Private Limited, 2016 3
 TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM

M 1 2 3 4 5 6 Materials Supplied:
The list below provides information about the materials supplied
Lane M : Lambda/Hind III Marker
in the kit. The components should be stored as suggested.
Lane 1 : Staphylococcus aureus 3A
Lane 2 : Bacillus amyloliquefaciens Quantity
Lane 3 : Bacillus globigi Materials 2115900031730 Store
Lane 4 : Klebsiella pneumonia (50 preps.)
Lane 5 : Neisseria sicca Proteinase K 1 ml -20°C
Lane 6 : Escherichia coli C RNase A 20 mg -20°C
Fig 1: DNA Purified from Lysozyme 100 mg -20°C
different Bacterial Strains Bacterial Lysis Buffer 5 ml 4°C
using GeneiPure™ Bacterial
DNA Purification Kit Elution Buffer 10 ml 4°C
Lysis Buffer I 10 ml RT
Lysis Buffer II 10 ml RT
M 1 2 3 4 5 6 7 M
Wash Buffer I 10 ml RT
Lane M : Quantum™ Low Range PCR (Concentrate)
Marker Wash Buffer II 7 ml RT
Lane 1-7 : RAPD of DNA Purified from (Concentrate)
TM
Mycobacterium smegmatis GeneiPure Columns 50 Nos. RT
using (different primers of
bacterial primer set). Collection Tubes 150 Nos. RT

Note: Room Temperature not to exceed 20-25°C.

Fig 2: RAPD profile of Materials Required but not provided:
Mycobacterium smegmatis
DNA purified using Bacterial Equipment : Microcentrifuge, Water Bath/Dry Bath, Vortex
DNA Purification Kit Reagent : Absolute ethanol (96-100%),
Glass Distilled Water (Sterile)
Other Requirements : 1.5 ml vials (Sterile)
 Genei Laboratories Private Limited, 2016 4  Genei Laboratories Private Limited, 2016 5
 TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM

Notes: Protocol:
• Read the entire procedure carefully prior to starting For Gram Positive Bacteria:
the experiment
1. Centrifuge 1-1.5 ml of bacterial culture at 6000 rpm for
• Upon storage at low temperatures some buffers like Lysis
10 minutes. Discard the supernatant.
Buffer I and Lysis Buffer II may form white precipitate.
Incubate the bottle at 50-70°C, till the precipitate 2. Resuspend the bacterial pellet in 100 µl of Bacterial Lysis
dissolves, mix well and use. Buffer. Incubate at 37°C for 30 minutes.
• Wash Buffer I and II are supplied as concentrates. Dilute Note: Add lysozyme to Bacterial Lysis Buffer just
required amount with 3 volumes of absolute ethanol just before use to a final concentration of 20 mg/ml. Mix
before use. Storage of diluted Wash Buffer is not thoroughly and store at 4°C.
recommended. 3. Add 180 µl of Lysis Buffer I. Add 20 µl of Proteinase K
• Set water bath/dry bath at 55°C and 70°C respectively for and mix thoroughly by vortexing. Incubate at 55°C for
use before starting experiment. 1-3 hours.
• Pre-warm only required amount of Elution Buffer at 70ºC Note: Increase the incubation time if sample is not
before use. completely lysed. Vortex the samples at intervals
• Reconstitute RNase A in sterile glass distilled water. for better lysis.
Please refer the table below for final concentration and 4. Add 200 µl of Lysis Buffer II mix thoroughly by vortexing
reconstitution details. After reconstitution store as and incubate at 70°C for 20 minutes.
aliquotes at -20°C. 5. Centrifuge at 10,000 rpm for 10 minutes and collect
supernatant in a sterile 1.5 ml vial.
Enzyme Final 612115900031730
6. Add 4 µl of RNase A (100 mg/ml) to the supernatant, mix
Conc. 50 preps.
by vortexing and incubate at room temperature for
RNase A 100 mg/ml 20 mg in 5-10 minutes.
200 µl 7. Add 200 µl of absolute ethanol and mix thoroughly by
vortexing.
8. Place the GeneiPureTM Column in a 2 ml Collection Tube
• Add lysozyme to Bacterial Lysis Buffer just before use to
and add the sample-ethanol mixture to the column.
a final concentration of 20 mg/ml. Mix thoroughly and store
at 4°C.
• All centrifugation steps are carried out at 11,000 rpm at
room temperature.

 Genei Laboratories Private Limited, 2016 6  Genei Laboratories Private Limited, 2016 7
 TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM

9. Centrifuge at 11,000 rpm for 5 minutes. Discard the 17. Place the GeneiPure Column in a fresh 1.5 ml vial and
Collection Tube with the flow through add 100 µl of pre-warmed Elution Buffer (70°C) to the
10. Dilute 1 volume of Wash Buffer I with 3 volumes of center of GeneiPure Column.
absolute ethanol just before use for the required number 18. Incubate at room temperature for 5 minutes and centrifuge
of DNA preparation. E.g. for one preparation, dilute for 1-2 minutes to elute the DNA.
125 µl of Wash Buffer I with 375 µl of absolute ethanol. 19. For maximum yield perform elution with 100 µl of additional
Mix thoroughly. Elution Buffer and repeat step 18.
11. Place the GeneiPureTM Column in a fresh 2 ml Collection 20. Store the eluted DNA at -20°C.
Tube and add 500 µl of diluted Wash Buffer I. Centrifuge
at 11,000 rpm for 1 minute. Discard the Collection Tube
with wash sample. For Gram Negative Bacteria:
12. Dilute 1 volume of Wash Buffer II with 3 volumes of absolute 1. Centrifuge 0.5-1 ml of culture at 6000 rpm for 10 minutes.
ethanol just before use for the required number of preps. 2. Proceed with Step 3 of Gram Positive Bacteria protocol.
E.g. for one preparation, dilute 125 µl of Wash Buffer II
with 375 µl of absolute ethanol. Mix thoroughly.
TM
13. Place the GeneiPure Column in a fresh 2 ml Collection For Mycobacterial Strain:
Tube and add 500 µl of diluted Wash Buffer II. Centrifuge 1. Centrifuge 0.5-1 ml of culture at 6000 rpm for 10 minutes.
at 11,000 rpm for 3 minutes. Discard wash fraction and 2. Resuspend bacterial pellet in 100 µl of Bacterial Lysis
retain the Collection Tube for next step. Buffer I containing 30 mg/ml lysozyme. Keep in boiling
Note: Following centrifugation, remove the water bath for 15-20 minutes.
GeneiPure TM Column from the Collection Tube 3. Proceed with Step 3 of Gram Positive Bacteria protocol.
carefully so that the Column does not come in
contact with the flow through.
14. Centrifuge the empty GeneiPureTM Column at 11,000 rpm
for 2 minutes. Discard Collection Tube.
15. Open the GeneiPureTM Column and place in a fresh sterile
1.5 ml vial. Incubate for 2 minutes at 70°C in a dry bath
to ensure complete removal of ethanol.
16. Take the required amount of Elution Buffer in a sterile
1.5 ml vial and pre-warm in a dry bath set at 70°C for
5 minutes. E.g. use 200 µl of Elution Buffer for one
preparartion.

 Genei Laboratories Private Limited, 2016 8  Genei Laboratories Private Limited, 2016 9
 TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM

Trouble Shooting: Observation: Suboptimal performance of DNA in

Observation: Low or no DNA recovery (Band intensity low downstream applications like PCR, RAPD, Restriction
or not seen) analysis etc

Possible Cause Comments Possible Cause Comments

Insufficient lysis Extend incubation time for Carry over ethanol Spin before eluting DNA for an
Proteinase K digestion with an additional 1 minute to remove traces
increase in the amount of of Wash Buffer
Proteinase K. Chaotropic salt in Dilute one volume of concentrated
eluted DNA Wash Buffer II with 4 volumes of
Excess amount of Reduce the amount of starting absolute ethanol.
starting material material to 0.5- 1 ml.

Old/overgrown Old/overgrown culture should be

culture avoided as it leads to degraded or
poor yield of DNA.
Incomplete DNA To increase DNA yield, elute with
elution buffer pre-warmed at 70ºC.

Observation: Loss of DNA in flow through and wash fractions

Possible Cause Comments
Over loading of Reduce the amount of starting
column with DNA material

Observation: RNA in the eluate

Possible Cause Comments
Insufficient RNase
Use a fresh vial of RNase
digestion

 Genei Laboratories Private Limited, 2016 10  Genei Laboratories Private Limited, 2016 11
 TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM

Ordering Information Flow Chart Gram Positive Bacteria

Lysis: 100 µl Bacterial Lysis
Product Size Cat # Buffer, incubate 37°C, 30 mins.
180 µl Lysis Buffer I +
TM 20 µl Proteinase K -55°C,
GeneiPure 1 EA 2115900031730 1-3 hours, vortex
Bacterial DNA Preparation Kit
(50 Preparations)
200 µl Lysis Buffer II incubate
70°C, 20 mins., vortex spin -
10,000 rpm for 10 mins. + To the
Other Related Products: supernatant add 4 µl, RNase A
Cat # Products incubate RT, 5 mins.
2115500031730 GeneiPure™ Mammalian Genomic DNA Prep Kit
2115200031730 GeneiPure™ Plasmid Purification Kit Binding: To lysed sample add
2115800031730 GeneiPure™ Yeast DNA Purification Kit 200 µl ethanol, spin 11,000 rpm
5 mins.
2115400031730 GeneiPure™ Gel Extraction Kit
2115900031730 GeneiPure™ Bacterial DNA Purification Kit
2115700031730 GeneiPure™ Plant Genomic DNA Purification Kit
2115600031730 GeneiPure™ Blood Genomic DNA Purification Kit
2150481001730 RNase A (DNase free) Wash I: 500 µl diluted
Wash Buffer I,
2150580201730 Proteinase K Solution 1 ml
spin 11,000 rpm 1 min.

Email:
Sales: sales@geneilabs.com
Customer Support: Wash II: 500 µl diluted
Wash Buffer II,
techsupport@geneilabs.com
spin 11,000 rpm 3 mins.

Elution: Required amount of

pre-warmed Elution Buffer (70°C)
incubate RT, 5 mins.,
spin 11,000 rpm, 2 mins.
DNA Fingerprinting Restriction Digestion
Hybridization PCR
Cloning

 Genei Laboratories Private Limited, 2016 12  Genei Laboratories Private Limited, 2016 13
 TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM TM
GeneiPure Bacterial DNA Purification Kit GeNeiTM

 Genei Laboratories Private Limited, 2016 14  Genei Laboratories Private Limited, 2016 15