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Bacteria Genomic DNA Extraction Kit 

Bacteria Genomic DNA Extraction Kit is designed for the rapid preparation of 5-20 µg genomic DNA from 109~2×109 viable bacterial cells (0.5 - 2.0 mL of culture).
Cat. No. Size Price Quantity  
TB50003A 50 preps USD 120
TB50003B 200 preps USD 380
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

Contents Volume
Buffer A 12 ml
Buffer B 12 ml
Buffer L 27 ml
Buffer W1 21 ml
Buffer W2 8 ml
Elution Buffer 12 ml
Proteinase K 1 ml
Spin Column 50 preps
Collection Tube 50 preps

Storage and Stability

2-8°C 12 months

Description

Bacteria Genomic DNA Extraction Kit is designed for the rapid preparation of 5-20 µg genomic DNA from 109~2×109 viable bacterial cells (0.5 - 2.0 mL of culture). The high-purity gDNA preparated by this kit usually has an OD260/OD280 between 1.8-2.0 and is highly suited for a variety of applications, including restriction analysis, sequencing, PCR, Southern blotting etc.

Bacteria Genomic DNA Extraction Kit is designed for the rapid preparation of genomic from both gram negative and gram positive cultures. The extraction process combines an initial lysis step using lysozyme and a subsequent proteolytic digestion step using proteinase K with a highly efficient process for binding bacterial gDNA to the surface of a Spin Column membrane. High-purity gDNA is eluted from the membrane after washing.

Important notes before starting

  1. For research use only. Not intended for human or diagnostic use.
  2. Buffer B and Buffer W1 contain strong protein denaturant and should be handled with care.
  3. Wear gloves, lab coat, etc., when handling the samples.
  4. All centrifugation steps should be carried out at room temperature (RT, 15-25℃).
  5. Avoid freeze/thaw cycle of bacterial samples.
  6. Ensure that no precipitates have formed in Buffer B and Buffer W1. If necessary, warm the solutions and mix well until the solutions become clear again.
  7. Add 7 ml and 32 ml ethanol (96-100%) into Buffer W1 and Buffer W2 respectively.
  8. Lysozyme is not privided with the kit and please prepare lysozyme buffer using Buffer L immediately before use, add lysozyme to 2 mg/ml.

Procedure

1. Sample Treatment
 
◆For Gram-negative bacteria:

Spin 1-2 ml of overnight bacterial culture at 9000g in a microcentrifuge for 1 min to pellet the cells. Discard supernatant.

◆For Gram-positive bacteria:
  1. Pellet the cell as for Gram-negative bacteria.
  2. Resuspend bacteria pellet in 500 μl lysozyme buffer (2 mg/ml), vortex to completely resuspend cell pellet and incubate at 37°C for 30-60 min, invert the tube every 20 min gently.
  3. Spin at 12000 rpm for 3 min to collect the cells and carefully remove as much of the supernatant as you can.
 
2. Add 180 μl Buffer A to the pellet and pipette up and down or vortex to resuspend the cells. Add 20 μl of proteinase K, mix by vortexing, and incubate at 55°C for 30 min.
 
3. If RNA-free genomic DNA is required, add 4 μl RNaseA (100 mg/ml) and mix the sample by inverting the tube 2-5 times. Incubate the mixture at room temperature for 2 minutes (Optional).
 
4. Add 200 μl Buffer B to the sample, mix by vortexing, and incubate at 70°C for 10 min to ensure the sample lysate is clear and homogenous.(White precipitates may form when Buffer B is added. They will not interfere with the procedure and will dissolve during the incubation at 70°C.)
 
5.Add 200 μl ethanol (96-100%), and mix well by vortexing. (A white precipitate may form on adding of ethanol. It is essential to apply all of the precipitate to the DNeasy Mini spin column. This precipitate does not interfere with the DNeasy procedure.)
 
6. Assemble spin column with one of the provided collection tubes and transfer the mixture (including the white precipitate) into the Spin Column and spin at 12000 rpm for 1 min, discard the flow-through and place the spin column into the collection tube.
 
7. Add 500 μl Buffer W1(Ensure ethanol (96-100%) has been added into the Spin Column,spin at 12000 rpm for 1 min, discard the flow-through and place the spin column into the collection tube.
 
8. Add 700 μl Buffer W2(Ensure ethanol(96-100%)has been added into the Spin Column,spin at 12000 rpm for 1 min, discard the flow-through and place the spin column into the collection tube.
 
9. Spin at 12000 rpm for 2 min and keep the Spin Column at room temperature for a few minutes to thoroughly dry the column.
 
10. Place the Spin Column in a new clean 1.5 ml centrifuge tube, add 50-200 μl pre-warmed Elution Buffer(65°C)directly to the center of the membrane,and incubate at room temperature for 2-5 min (The volume of Elution buffer should be more than 50μl and distilled water (pH > 7.0) also can be used to elute gDNA).
 
11. Spin at 12000 rpm for 2 min to elute DNA. (The yield can be improved by pipetting the elution back onto the column and repeating step11.)

Troubleshooting

The yield of genomic DNA is low

This may caused by incomplete lysis of cells or the sample is old/overgrown.
  1. Incomplete lysis of cells. Try to reduce the amount of bacterial cells and too many cells may slao results in clogging the spin column. Increase the lysozyme incubation time for gram positive strains. Extend the incubation time of Proteinase K digestion.
  2. The sample is old/overgrown. It may be necessary to use bacterial cultures before they reach maximum density. If users need to process the preparation another day; pellet the cells, remove the culture medium and store the pellet at -70°C.
  3. Make sure ethanol (96-100%) had been added into Buffer W1 and Buffer W2.

Downstream application failure with gDNA eluate

Keep the Spin Column at room temperature for a few minutes to dry thoroughly the column before adding elution buffer and the elution buffer should be added directly to the center of the membrane.

Datasheet Download

Bacteria Genomic DNA Extraction Kit

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