Instruction Manual

Ver. 02.10.17
For Research Use Only

Presto™ Mini gDNA Bacteria Kit

GBB004 (4 Preparation Sample Kit)
GBB100/101 (100 Preparation Kit)
GBB300/301 (300 Preparation Kit)

Advantages
Sample: up to 1 x 109 Gram (+) positive and Gram (-) negative bacterial cells, 200 µl of blood and
biological fluids such as plasma, urine etc.
gDNA Yield: up to 40 µg from 1 x 109 Escherichia coli and up to 15 µg from 1 x 109 Bacillus subtilis
Convenient: includes Gram+ Buffer for preparing lysozyme solutions and to speed up sample
preparation
Format: genomic DNA spin columns (sterilised to remove bacteria contamination)
Time: within 30 minutes
Elution Volume: 30-200 μl
Kit Storage: dry at room temperature (15-25ºC), Lysozyme is shipped at room temperature and
should be stored at -20ºC for extended periods

Table of Contents
Introduction...................................................................................................................................................... 2
Quality Control................................................................................................................................................. 2
Kit Components............................................................................................................................................... 2
Safety Measures............................................................................................................................................... 3
Quick Protocol Diagram................................................................................................................................ 3
Protocol Procedure......................................................................................................................................... 4
Troubleshooting.............................................................................................................................................. 6
Test Data............................................................................................................................................................. 7
Related Products............................................................................................................................................. 7

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Introduction
The Presto™ Mini gDNA Bacteria Kit is optimized for genomic and viral DNA purification from Gram (-)
negative and Gram (+) positive bacterial cells, whole blood and biological fluids. Gram+ Buffer, when
combined with Lysozyme, will efficiently lyse bacterial cell walls consisting of the peptidoglycan layer.
Proteinase K and chaotropic salt are used to further lyse cells and degrade protein, allowing DNA to
easily bind to the glass fiber matrix of the spin column. Contaminants are removed using a Wash Buffer
(containing ethanol) and the purified genomic DNA is eluted by a low salt Elution Buffer, TE or water.
Phenol/chloroform extraction or alcohol precipitation is not required and the purified genomic DNA is
ready for use in a variety of downstream applications.

Quality Control
The quality of the Presto™ Mini gDNA Bacteria Kit is tested on a lot-to-lot basis by isolating DNA from
Escherichia coli (1×109) culture harvested by centrifugation at 16,000 x g for 1 minute. 5 µl from a 100 µl
eluate of purified DNA is analyzed by electrophoresis on a 1% agarose gel.

Kit Components
Component GBB004 GBB100 GBB101 GBB300 GBB301
Gram+ Buffer 2 ml 30 ml 30 ml 75 ml 75 ml
GT Buffer 1.5 ml 30 ml 30 ml 75 ml 75 ml
GB Buffer 2 ml 40 ml 40 ml 75 ml 75 ml
W1 Buffer 2 ml 45 ml 45 ml 130 ml 130 ml
1
Wash Buffer 1 ml 25 ml 25 ml 50 ml 50 ml
(Add Ethanol) (4 ml) (100 ml) (100 ml) (200 ml) (200 ml)
2
Lysozyme 8 mg 110 mg N/A 250 mg N/A
3
Proteinase K 1 mg 11 mg x 2 11 mg x 2 65 mg 65 mg
(Add ddH2O) (0.1 ml) (1.1 ml) (1.1 ml) (6.5 ml) (6.5 ml)
Elution Buffer 1 ml 30 ml 30 ml 75 ml 75 ml
GD Columns 4 100 100 300 300
2 ml Collection Tubes 8 200 200 600 600
1
Add absolute ethanol (see the bottle label for volume) to Wash Buffer then mix by shaking for a few
seconds. Check the box on the bottle. Be sure and close the bottle tightly after each use to avoid ethanol
evaporation.
2
Lysozyme should be stored at -20ºC for extended periods.
3
Add ddH2O (see the bottle label for volume) to Proteinase K then vortex to ensure Proteinase K is
completely dissolved. Check the box on the bottle. Once it is dissolved completely, centrifuge for a few
seconds to spin down the mixture. For extended periods, the ddH2O and Proteinase K mixture should be
stored at 4ºC. Use only fresh ddH2O as ambient CO2 can quickly cause acidification.

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During the procedure, always wear a lab coat, disposable gloves, and protective
goggles.

Q u i c k P r o t o c o l Diagram

Sample preparation and cell lysis of bacteria, whole blood and biological fluids

DNA binding to membrane while contaminants remain suspended

Wash (removal of contaminants while DNA remains bound to membrane)

Elution of pure genomic DNA which is ready for subsequent reactions

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PrestoTM Mini gDNA Bacteria Kit Protocol

Please read the entire instruction manual prior to starting the Protocol Procedure.
IMPORTANT BEFORE USE!
1. Add absolute ethanol (see the bottle label for volume) to Wash Buffer then mix by shaking for a few
seconds. Check the box on the bottle. Be sure and close the bottle tightly after each use to avoid ethanol
evaporation.
2. Add ddH2O (see the bottle label for volume) to Proteinase K then vortex to ensure Proteinase K is
completely dissolved. Check the box on the bottle. Once it is dissolved completely, centrifuge for a few
seconds to spin down the mixture. For extended periods, the ddH2O and Proteinase K mixture should be
stored at 4ºC. Use only fresh ddH2O as ambient CO2 can quickly cause acidification.

Additional Requirements
absolute ethanol, microcentrifuge tubes, pipette tips, RNase A (50 mg/ml), 15 ml centrifuge tube (Gram
positive bacteria only)

Protocol Procedure
1. Sample Preparation
Gram (-) Negative Bacteria
Transfer bacterial cells (up to 1 x 109) to a 1.5 ml microcentrifuge tube. Centrifuge for 1
minute at 14-16,000 x g then discard the supernatant. Add 180 µl of GT Buffer then
re-suspend the cell pellet by vortex or pipette. Add 20 µl of Proteinase K (make sure ddH2O
was added). Incubate at 60ºC for at least 10 minutes. During incubation, invert the tube every
3 minutes. Proceed with step 2 Lysis.

Gram (+) Positive Bacteria

Transfer bacterial cells (up to 1 x 109) to a 1.5 ml microcentrifuge tube. Centrifuge for 1
minute at 14-16,000 x g then discard the supernatant. Transfer the required volume of
Gram+ Buffer (200 µl/sample) to a 15 ml centrifuge tube. Add Lysozyme (0.8 mg/200 µl) to
Gram+ Buffer (in the 15 ml centrifuge tube) then vortex to completely dissolve the
Lysozyme. Transfer 200 μl of Gram+ Buffer (make sure Lysozyme was added) to the
sample in the 1.5 ml microcentrifuge tube then re-suspend the pellet by vortex or pipette.
Incubate at 37ºC for 30 minutes. During incubation, invert the tube every 10 minutes. Add 20
µl of Proteinase K (make sure ddH2O was added) then mix by vortex. Incubate at 60ºC for
at least 10 minutes. During incubation, invert the tube every 3 minutes. Proceed with step 2
Lysis.

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Whole Blood

Transfer 200 µl of blood to a 1.5 ml microcentrifuge tube. Add 20 µl of Proteinase K (make

sure ddH2O was added) then mix by vortex for 10 seconds. Incubate at 60ºC for at least 10
minutes. During incubation invert the tube every 3 minutes. Proceed with step 2 Lysis.

Biological Fluids

Transfer 1 ml of biological fluid to a 1.5 ml microcentrifuge tube. Centrifuge for 5 minutes at

14-16,000 x g then discard the supernatant.
NOTE: When using more than 1 ml of biological fluid, repeat the centrifuge step.
Add 200 µl of GT Buffer then re-suspend the pellet by vortex or pipette. Add 20 µl of
Proteinase K (make sure ddH2O was added) then mix by vortex. Incubate at 60ºC for at
least 10 minutes. During incubation invert the tube every 3 minutes. Proceed with step 2
Lysis.

2. Lysis
Add 200 µl of GB Buffer to the sample and mix by vortex for 10 seconds. Incubate at 70ºC
for at least 10 minutes to ensure the sample lysate is clear. During incubation, invert the tube
every 3 minutes. At this time, pre-heat the required Elution Buffer (200 μl per sample) to 70ºC
(for step 5 DNA Elution).

Optional RNA Removal Step

Following 70ºC incubation, add 5 μl of RNase A (50 mg/ml) to the clear lysate then shake
vigorously. Incubate at room temperature for 5 minutes.

3. DNA Binding
Add 200 µl of absolute ethanol to the sample lysate and mix IMMEDIATELY by shaking
vigorously. If precipitate appears, break it up as much as possible with a pipette. Place a GD
Column in a 2 ml Collection Tube. Transfer mixture (including any insoluble precipitate)
to the GD Column then centrifuge at 14-16,000 x g for 2 minutes. Discard the 2 ml Collection
Tube containing the flow-through then place the GD Column in a new 2 ml Collection Tube.

4. Wash
Add 400 µl of W1 Buffer to the GD Column. Centrifuge at 14-16,000 x g for 30 seconds then
discard the flow-through. Place the GD Column back in the 2 ml Collection Tube. Add 600 µl
of Wash Buffer (make sure ethanol was added) to the GD Column. Centrifuge at
14-16,000 x g for 30 seconds then discard the flow-through. Place the GD Column back in
the 2 ml Collection Tube. Centrifuge again for 3 minutes at 14-16,000 x g to dry the column
matrix.

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5. Elution
Standard elution volume is 100 μl. If less sample is to be used, reduce the elution volume (30-50 μl) to
increase DNA concentration. If higher DNA yield is required, repeat the DNA elution step to increase DNA
recovery and the total elution volume to approximately 200 μl.

Transfer the dried GD Column to a clean 1.5 ml microcentrifuge tube. Add 100 μl of
pre-heated Elution Buffer1, TE Buffer2 or water3 into the CENTER of the column matrix. Let
stand for at least 3 minutes to allow Elution Buffer, TE Buffer or water to be completely
absorbed. Centrifuge at 14-16,000 x g for 30 seconds to elute the purified DNA.
Ensure that Elution Buffer (10 mM Tris-HCl, pH8.5 at 25ºC) is added into the center of the GD Column
1

matrix and is completely absorbed.

2
Using TE (10 mM Tris-HCl, 1 mM EDTA, pH8.0) for elution is beneficial as EDTA preserves DNA for long
term storage. However, EDTA will affect PCR and other sensitive downstream applications. Ensure that
TE is added into the center of the GD Column matrix and is completely absorbed.
3
If using water for elution, ensure the water pH is between 7.0 and 8.5. ddH2O should be fresh as ambient
CO2 can quickly cause acidification. Ensure that water is added into the center of the GD Column matrix
and is completely absorbed. DNA eluted in water should be stored at -20ºC to avoid degradation.

Troubleshooting
Low Yield
Incomplete buffer preparation.
1. When extracting genomic DNA from Gram (+) positive bacteria, add Lysozyme to Gram+
Buffer immediately prior to use.
2. Add absolute ethanol (see the bottle label for volume) to Wash Buffer then mix by shaking
for a few seconds. Check the box on the bottle. Be sure and close the bottle tightly after each
use to avoid ethanol evaporation.
3. Add ddH2O (see the bottle label for volume) to Proteinase K then vortex to ensure
Proteinase K is completely dissolved. Check the box on the bottle. Once it is dissolved
completely, centrifuge for a few seconds to spin down the mixture. For extended periods, the
ddH2O and Proteinase K mixture should be stored at 4ºC. Use only fresh ddH2O as ambient
CO2 can quickly cause acidification.
Incomplete cell lysis.
Reduce the amount of starting material or separate into multiple tubes. Make sure bacteria
cells were completely homogenized in GT Buffer or Gram+ Buffer. If extracting genomic DNA
from Gram (+) positive bacteria, make sure Lysozyme is added to Gram+ Buffer prior to use.

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Incorrect DNA elution step.

Ensure that Elution Buffer, TE or water is added into the CENTER of the GD Column matrix
and is completely absorbed. Use pre-heated Elution Buffer, TE, or water (60~70ºC). If using
water for elution, ensure the water pH is between 7.0 and 8.5. ddH2O should be fresh as
ambient CO2 can quickly cause acidification. Repeating the elution step will increase yield.
Repeating the elution step using the eluate only will increase DNA concentration.

Eluted DNA Does Not Perform Well In Downstream Applications

Residual ethanol contamination.
Following the wash step, dry the GD Column with additional centrifugation at 14-16,000 x g
for 5 minutes to ensure the GD Column membrane is completely dry.

Presto™ Mini gDNA Bacteria Kit Functional Test Data

Figure 1. Genomic DNA (approximately 30 kb) was extracted using the Presto™ Mini gDNA
Bacteria Kit. An Escherichia coli (1×109) culture (OD600=2, 1 ml) was harvested by centrifugation
at 16,000 x g for 1 minute. A 5 µl aliquot of purified genomic DNA from a 100 µl eluate was
analyzed by electrophoresis on a 1% agarose gel. M = Geneaid 1 Kb DNA Ladder
Test DNA Yield 260/280 260/230
1 38.16 µg 2.05 2.26
2 33.88 µg 2.05 2.27
3 39.02 µg 2.07 2.29
M 1 2 3

Figure 2. Genomic DNA (approximately 30 kb) was extracted using the Presto™ Mini gDNA
Bacteria Kit. A Bacillus subtilis (1×109) culture (OD600=2, 1 ml) was harvested by centrifugation
at 16,000 x g for 1 minute. A 5 µl aliquot of purified genomic DNA from a 100 µl eluate was
analyzed by electrophoresis on a 1% agarose gel. M = Geneaid 1 Kb DNA Ladder
Test DNA Yield 260/280 260/230
1 12.28 µg 1.96 2.28
2 12.08 µg 1.97 2.30
3 12.56 µg 1.96 2.29
M 1 2 3

Related DNA Extraction Products

Genomic DNA Extrac�on and Purifica�on
Product Package Size Catalogue Number
Genomic DNA Mini Kit (Blood/Cultured Cell) 100/300 preps GB100/101/300/301
Genomic DNA Mini Kit (Tissue) 50/100/300 preps GT050/100/300
gSYNC™ DNA Extrac�on Kit 50/100/300 preps GS050/100/300
Genomic DNA Mini Kit (Plant) 100 preps GP100
Genomic DNA Maxi Kit (Plant) 10/25 preps GPM10/25
GENEzol™ DNA Reagent Plant 100/200 rxns GR100/200
Presto™ Mini gDNA Yeast Kit 100/300 preps GBY100/300
Presto™ Mini gDNA Bacteria Kit 100/300 preps GBB100/300
Geneius™ Micro DNA Extrac�on Kit 100/300 preps GMB100/300
Presto™ Buccal Swab gDNA Extrac�on Kit 100/300 preps GSK100/300
For additional product information please visit www.geneaid.com. Thank you!

Geneaid Biotech Ltd.

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