Procedure Isolation and Purification of Total Genomic DNA from a bacteria.

This will be a small scale isolation and purification, providing DNA of sufficient purity for use as template in a PCR reaction.

wear goggles and gloves

discard reagents and tubes in labeled waste containers

1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.

2. Centrifuge at 15,000 × g for 2 minutes to pellet the cells.

Place your tube opposite that of another student to balance to rotor.

Do not initiate a spin cycle until the rotor is fully loaded; this minimizes the total number of runs required.

3. Transfer the supernatant back into the culture tube it came from and discard this culture tube as biohazard waste.

Carefully remove as much of the supernatant as you can without disturbing the cell pellet. The pellet may be on the side of the tube, not squarely on the bottom.

4. Resuspend the cell pellet in 600µl of Lysis Solution (LS) and then incubate at 80°C for 5 minutes to lyse the cells.

Gently pipet until the cells are thoroughly resuspended.

LS contains the anionic detergent sodium dodecyl sulphate (SDS).

5. Cool the tube contents to room temperature.

Do not rely on temperature equilibration with ambient air. Place the tube in a room temperature water bath for several minutes.

6. Add 3µl of RNase solution to the cell lysate. Invert the tube 2–5 times to mix.

7. Incubate at 37°C for 15–60 minutes to digest RNA. Cool the sample to room temperature.

8. Add 200 µl of Protein Precipitation Solution (PPS) to the RNase-treated cell lysate. Vortex vigorously at high speed for 20 seconds to mix.

9. Incubate the sample in an ice/water slurry for 5 minutes.

10. Centrifuge at 15,000 × g for 3 minutes.

11. Transfer the supernatant (≤800 µl) containing the DNA to a clean 1.5ml microcentrifuge tube containing 600µl of room temperature isopropanol (IPA).

Be sure that you don’t suck up and transfer any of the grungy precipitate.

12. Gently mix by inversion. The DNA should come out of solution as visible thread-like strands.

13. Centrifuge at 15,000 × g for 2 minutes.

14. Carefully pour off the supernatant (do not pipette) and drain the tube on clean absorbent paper.

The DNA pellet may not be visible.

Do not allow the DNA pellet to dry out.

15. Add 600µl of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet.

16. Centrifuge at 15,000 × g for 2 minutes.

17. Carefully pour off the ethanol (do not pipette) and drain the tube on clean absorbent paper.

18. Allow the pellet to air-dry for 10–15 minutes.

You want to evaporate as much of the ethanol as possible without letting the DNA pellet completely dry.

19. Add 100µl of DNA Rehydration Solution (RH) to the tube and rehydrate the DNA by incubating at 65°C for 1 hour.

Periodically mix the solution by gently tapping the tube.

Alternatively, rehydrate the DNA by incubating the solution overnight at room temperature or at 4°C, preferably on a low speed shaker.

20. Store the DNA at 2–8°C