Genomic DNA Purification Protocol

Modification to the salting out procedure of Miller, S.A., et al., Nucleic Acids Research, 16, 1215 (1988). Modification by Martha Feller

1. Resuspend the cell pellet in 9 ml of TE9 in a 50 ml conical tube. Add 1 ml of 10% SDS and invert to mix. Add 0.25 ml proteinase K (20 mg/ml) and invert to mix.

2. Incubate at 48C overnight.

3. Add 2.5 ml saturated NaCl (6M), cap and shake vigorously for 15 seconds, then centrifuge at 2500 rpm for 30 minutes. There will be a protein pellet at the bottom of the tube.

Then, either:

4. A. Pour the supernatant into another 50 ml conical and add 2 volumes of room temperature ethanol. Invert several times to precipitate DNA, and remove DNA strands by fishing out with a pasteur pipet

OR (for those who prefer, as I do)

B. Pour the supernatant into a 50 or 100 ml glass beaker, add two volumes of room temperature, and spool out the DNA onto a pasteur pipet whose end has been heat-sealed. Squeeze out the excess liquid by rolling the spooled DNA against the side of the beaker.

5. Put the DNA strands into a 1.5 ml microfuge tube containing 1 ml of TE pH 7.5. (Volume can be increased or decreased according to DNA amount). If you have spooled the DNA, you can either scrape it off the pipet as it rehydrates, (rolling the pipet between the fingers helps get the DNA off into the buffer). Or, break off the pipet tip and leave it in the tube until later. Allow to stand at 37C 2 hours (at least).

Solutions:

• TE9 500 mM Tris pH9.0 20 mM EDTA 10 mM NaCl
• TE 7.5 10 mM Tris pH 7.5 0.2 mM EDTA