Bacterial Genomic DNA Extraction

DNA extraction from unknown bacterial colonies extracted from biofilms and planktonic cultures grown on a variety of media types. This protocol is based of protocols from Monika Schwering and MP Bio's DNA Spin Kit extraction protocol.

Materials:

• MP Bio FastPrep bead beater
• Micro-centrifuge
• Heating Block

MP Bio FastDNA SPIN Kit (cat # 6540-600)

• Lysing Matrix A Tubes
• DNA catch tubes
• 2.0 mL eppendorf tubes
• 1.5 mL eppendorf tubes
• CLS-TC buffer
• DNA binding matrix
• SEWS-M diluted with ethanol
• DES water
• Lysozyme (EC 3.2.1.17)
• Lysis buffer (20 mM Tris, 2 mM EDTA, 1% Triton X-100, adjusted to pH 8.0)
• Proteinase K

Procedure:

1. Prepare 5mL overnight cultures of each organism to be extracted in a 15mL Falcon tube. Incubate overnight in a shaker at the required growth temperature and rotation speed for the organism of interest.
2. Centrifuge the overnight cultures at 3000rpm for 10 minutes. Dump the supernatant and use a 200 µL pipette tip to remove any excess supernatant remaining in the tube.
3. Prepare the lysis buffer () with 20 mg/mL concentration of lysozyme.

a. First, calculate the total volume of lysis buffer needed for the experiment:

i. (# of samples + 0.5 for pipetting error) * 0.200 mL of lysis buffer per sample

ii. ie: (12 samples + 0.5) * 0.200 mL = 2.5 mL of lysis buffer

b. Next, calculate the quantity of lysozyme required to achieve a concentration of 20 mg/mL of lysozyme in the lysis buffer:

i. Total volume of lysis buffer * 20 mg/mL of lysozyme

ii. ie: 2.5 mL * 20 mg/mL = 50 mg of lysozyme

4. Add 200 µL of lysis buffer to each samples tube and vortex each sample to re-suspend the mixture. Place the tubes on a shaker in a 37ºC incubator for 30 minutes.
5. Assemble one lysing matrix A tube for each sample and place 880 µL of CLS-TC and 20 µL of proteinase K in each tube.
6. Set a heating block to 55ºC.
7. Remove the samples from the shaker in the 37ºC incubator. Vortex each samples to re-suspend any precipitate and pipette all of the sample (using a pipette set at 300 µL) into a separate lysing matrix A tube.
8. Bead beat the samples for 40 seconds at speed 6.0.
9. Place the samples on the 56ºC heating block for 30 minutes.
10. Spin the samples for 10 minutes at 14,000xg.
11. Prepare one 2.0 mL eppendorf tube for each sample and pipette 800 µL of DNA binding matrix into each tube. Then pipette, 800 µL of the supernatant from each lysing matrix A tube into the corresponding tube. Ensure that the precipitate is not disturbed during this process.
12. Invert the tubes for 5 minutes by hand.
13. Gently invert each sample tube to ensure that there is no precipitate and pipette 800 µL (1/2 of the sample) into a catch tube.
14. Spin the catch tubes for 1 minute at 14,000xg
15. Empty each catch tube and transfer the remaining ~800 µL of sample into the corresponding catch tubes.
16. Spin the catch tubes for 1 minute at 14,000xg. Empty the catch tubes.
17. Add 500 µL of SEWS-M/ethanol to each catch tube and pipetting up and down to re-suspend the pellet
18. Spin the catch tubes for 1 minute at 14,000xg. Empty the catch tubes.
19. Spin the catch tubes again for 2 minutes at 14,000xg to dry out the pellets.
20. Replace the recovery tubes with fresh 2.0 mL eppendorf tubes with final storage labeling.
21. Add 100 µL of DES water and re-suspend the pellets by pipetting up and down. Incubate the tubes at 55ºC for 30 minutes in a heating block.
22. Spin down the tubes for 2 minutes at 14,000xg. Keep the liquid and tube. Dispose of the filter column.
23. Storage: place the tubes into a -20ºC freezer.