Bacterial Genomic DNA Extraction Kit

This kit enables rapid extraction and purification of bacterial genomic DNA through a specially optimized bacterial lysis buffer system coupled with centrifugal adsorption columns that specifically bind DNA. This kit is suitable for the extraction of genomic DNA from a wide range of Gram-negative bacteria, as well as the extraction of genomic DNA from the majority of Gram-positive bacteria using a combination of lysozyme and lysis buffer system. The genomic DNA obtained is characterized by high purity and high integrity, and can be directly used in a variety of molecular biology experiments such as subsequent PCR amplification, Southern hybridization and library construction.

Lysozyme, Proteinase K, RNase A are shipped with wet ice and stored at -20°C; the rest of the reagents are shipped and stored at room temperature; valid for 12 months.

1. If a precipitate has formed in Buffer BGL1, heat it at 65°C until the precipitate has fully dissolved, and use it after it returns to room temperature.

2. Before use, add 18 mL of absolute ethanol to Buffer BPD, add 56 mL of absolute ethanol to Buffer PW.

1. Add 1-5 mL of fresh overnight bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 1 minute, and discard the supernatant. (If there is more bacterial solution, collect the bacterial precipitate into a centrifuge tube through multiple centrifugations.

Note: For Gram-positive bacteria, lysozyme solution should be added to break the cell wall. The method is as following: add 130 µL Lysozyme Buffer, 50 µL Lysozyme, vortex shaking until the bacteria are thoroughly suspended, water bath at 37°C for 60 min, mixing every 10 min inverted.

2. Add 200 μL of Buffer BGL1 to the centrifuge tube and mix by vortexing and shaking.

3. Add 20 μL of Proteinase K and 10 µL of RNase A to mixture, mix well by pipetting or vortexing and incubate at 56℃ for 20 min. The solution should appear clear, and clarify.

Note: After incubation, the solution should become clear. If not, extend the incubation time to 30 minutes, and mix by pipetting once every 10 minutes.

4. Add 200 μL of Buffer BGL2 and 200 μL of absolute ethanol to the centrifuge tube, mix thoroughly upside and down, and white precipitate may be produced at this step.

5. Place the DNA Spin Columns into the Collection Tubes, transfer all the mixture to the DNA Spin Columns.

6. Centrifuge at 12,000 rpm for 30 seconds, discard the flow-through., and put the DNA Spin Columns back into the collection tube.

7. Add 500 μL Buffer BPD to the DNA Spin Columns, centrifuge at 12,000 rpm for 30 seconds, discard the flow-through.

8. Add 600 μL Buffer PW to the DNA Spin Columns (please add Buffer PW along the wall of the tube, it helps to rinse the residual salt on the wall of the tube), centrifuge at 12,000 rpm for 30 seconds, discard the flow-through.

9. Repeat Step 8.

10. Place the DNA Spin Column in a Collection Tube and centrifuge at 12,000 rpm for 2 min to remove as much residue as possible.

11. Put the DNA Spin Columns into a new 1.5 mL centrifuge tube, stand at room temperature for 5-10 minutes to allow the residual ethanol from the DNA Spin Column to completely evaporate.

12. Add 50-100 μL Buffer TE or nuclease-free water to the membrane center of DNA Spin Column and leave for 5 min at room temperature. (Pre-heat the Buffer TE or nuclease-free water at 65℃ can improve elution efficiency).

13. Centrifuge at 12,000 rpm for 2 minutes to collect DNA. If more yield is needed, the flow-through can be re-added into DNA Spin Column and let it stand for 5 minutes at room temperature, centrifuge at 12,000 rpm for 2 minutes and collect the DNA again.

1. Please read the Product Manual carefully before use.

2. Use fresh experimental material to ensure the yield and integrity of the genomic DNA obtained by extraction.

3. The starting amount of extracted bacteriophage should never be too much and should be sufficiently lysed, otherwise it may affect the yield and purity of genomic DNA.

4. For your safety and health, please wear safety glasses, gloves, or protective clothing.

For Research Use Only!