Bactierial DNA Isolation Procedure

Instructions for Use
Version: 1.0.1
Bacteria Genomic DNA Kit
Catalog No.: abx098080
Size: 50 rxns
Storage: Store RNase A at -20 °C for up to two years, and the other components at room temperature (15-25 °C) for
up to one year.
Introduction
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Abbexa's Bacteria Genomic DNA Kit uses lysozyme and moderate lysis buffer to lyse cells. Proteinase K is used for
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protein digestion and RNase A used for RNA digestion. DNA is specifically bound to a silica-based column in a
hypersaline environment, and DNA is eluted by a low salt and high pH solution. This kit is suitable for isolating high
quality genomic DNA from gram-positive and gram-negative bacteria. The isolated genomic DNA is suitable for PCR,
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restriction enzyme digestion and southern blotting.
Kit Components
Reagent
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• Resuspension Buffer: 12 ml • 96-100% ethanol
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• Lysis Buffer: 6 ml • 70% ethanol
• Binding Buffer: 10 ml • Lysozyme
• Clean Buffer: 55 ml • Glass beads
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• Wash Buffer: 12 ml • Pipettes and pipette tips

• Elution Buffer: 25 ml • Centrifuge and centrifuge tubes
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• Proteinase K (20 mg/ml): 1 ml • Homogenizer

• RNase A (10 mg/ml): 1 ml • Water bath or incubator
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• Genomic Spin Columns with Collection Tubes: 50
Protocol
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Reagent Preparation
• Working Binding Buffer solution: Dilute the Binding Buffer with 96-100% ethanol to a ratio of 2:3 (i.e. add 10
ml of Binding Buffer into 15 ml of 96-100% ethanol to form 25 ml of Working Binding Buffer solution).
• Working Wash Buffer solution: Dilute the Wash Buffer with 96-100% ethanol to a ratio of 1:4 (i.e. add 12 ml of
Wash Buffer into 48 ml of 96-100% ethanol to form 60 ml of Working Wash Buffer solution).
• Working Lysozyme solution: Dissolve 4 mg of lysozyme in 200 µl of Resuspension Buffer.
Sample Preparation
Samples should have a gram-positive or gram-negative bacteria cell count of ≤ 109. Samples which contain a bacteria
cell count above this may result in incomplete lysis.
Abbexa UK • Abbexa US • Abbexa NL

www.abbexa.com • [email protected]
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Instructions for Use
Version: 1.0.1
• Lysis of gram-negative bacteria:
1. Transfer 1 ml of overnight culture gram-negative bacteria to 1.5 ml centrifuge tube, and centrifuge at 12,000
× g for 1 minute. Discard the supernatant.
2. Add 100 µl of Lysis Buffer and 20 µl of Proteinase K to the pellet.
3. Resuspend the bacteria by vortexing. Incubate at 55 °C for 15 minutes. The solution should be clear after
incubation, if not, extend the incubation time to 30 minutes, vortexing every 5 minutes.
• Lysis of gram-positive bacteria:
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1. Transfer 1 ml of overnight culture gram-negative bacteria to 1.5 ml centrifuge tube, and centrifuge at 12,000
× g for 1 minute. Discard the supernatant.
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2. Gram-positive coccus only: Resuspend the pellet in 500 µl of 70% ethanol, then put on ice for 20 minutes.
Centrifuge at 10,000 × g for 1 minute, then discard the supernatant.
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Actinomyces only: Use glass beads to break the hyphae clump. Centrifuge at 10,000 × g for 1 minute then
discard the supernatant.
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Add 200 µl of Working Lysozyme solution (dissolved in Resuspension Buffer) to the tube. Incubate at 37 °C
for at least 60 minutes. The incubation time can be extended to up to 3 hours, depending on the amount of
bacteria. Centrifuge at 10,000 × g for 1 minute, then discard the supernatant.
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4. Add 100 µl of Lysis Buffer and 20 µl of Proteinase K to the pellet. Resuspend the bacteria by vortexing.
Incubate at 55 °C for 15 minutes. The solution should be clear after incubation, if not, extend the incubation
time to 30 minutes, vortexing every 5 minutes.
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Assay Procedure
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Use sterile tubes and pipette tips to avoid DNase contamination.
1. Add 20 µl of RNase A to the tube. Mix and allow to stand at room temperature for 2 minutes.
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2. Add 400 µl of Working Binding Buffer solution (with ethanol) to the tube and vortex for 30 seconds. A white
precipitate or transparent gelatinous matter may be visible at this stage, this does not affect DNA extraction.
3. Transfer the entire contents of the tube to a spin column. Centrifuge at 12,000 × g for 30 seconds. Discard the
flow-through.
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4. Add 500 µl of Clean Buffer to the column. Centrifuge at 12,000 × g for 30 seconds. Discard the flow-through.
5. Repeat Step 4 above once more.
6. Add 500 µl of Working Wash Buffer solution (with ethanol) to the column. Centrifuge at 12,000 × g for 30 seconds.
Discard the flow-through.
7. Repeat Step 6 above once more.
8. Centrifuge at 12,000 × g for 2 minutes to remove residual Working Wash Buffer solution.
9. Place the spin column in a sterile 1.5 ml microcentrifuge tube. Add 50-200 µl of Elution Buffer (preheated to 65
°C to increase DNA yield) or sterile distilled water (pH > 7.0) to the center of the column. Allow to stand at room
temperature for 2 minutes. Centrifuge at 12,000 × g for 1 minute to elute the genomic DNA.
10. Repeat Step 9 above once more. The purified DNA can be stored at -20 °C for long-term storage.
Abbexa UK • Abbexa US • Abbexa NL

www.abbexa.com • [email protected]
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Instructions for Use

Bacteria Genomic DNA Kit

Catalog No.: abx098080

• Wash Buffer: 12 ml • Pipettes and pipette tips

• Proteinase K (20 mg/ml): 1 ml • Homogenizer

• Genomic Spin Columns with Collection Tubes: 50

• Working Lysozyme solution: Dissolve 4 mg of lysozyme in 200 µl of Resuspension Buffer.

Abbexa UK • Abbexa US • Abbexa NL

• Lysis of gram-negative bacteria:

2. Add 100 µl of Lysis Buffer and 20 µl of Proteinase K to the pellet.

• Lysis of gram-positive bacteria:

Use sterile tubes and pipette tips to avoid DNase contamination.

Abbexa UK • Abbexa US • Abbexa NL

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