Posted 30 June 2009 - 07:59 AM
After a phenol extraction, we will extract again with phenol:chloroform:isoamyl alcohol, and then extract with chloroform until there is no white interface. Here is our protocol for genomic DNA:
1. Grow cells overnight in 5 ml of appropriate media.
2. Fill one or more 1.5 ml microcentrifuge tubes with the overnight culture.
3. Pellet the cells by centrifugation in a table-top microfuge.
4. Decant the supernatant, and resuspend the pellet in 600 µl TE buffer.
5. Add 17 µl of 20% SDS (thus 0.5% v/v).
6. Add 2 µl of a 20 mg/ml stock of proteinase K (thus 50 µg/ml).
7. Mix by inverting several times, and incubate at 56°C for two or more hours†.
8. Add 600 µl buffer-saturated phenol to the tube. Mix by shaking.
9. Centrifuge for 5 minutes in a bench-top centrifuge.
10. Transfer the aqueous (top) layer to a fresh tube, avoiding the white interface layer. Discard the organic (bottom) phase.
11. Add 100 µl TE to the aqueous phase ‡
12. Add 600 µl phenol:chlorofom:isoamyl alcohol (25:24:1).
13. Mix by inverting several times, then separate the phases by centrifuging for 5 minutes.
14. Transfer the aqueous (top) layer to a fresh tube, avoiding the white interface layer. Discard the organic (bottom) phase.
15. Add 100 µl TE to the aqueous phase.
16. Add 600 µl chloroform.
17. Mix by inverting several times, then separate the phases by centrifuging for 5 minutes.
18. Repeat the chloroform extraction (above three steps) until no white interface remains §.
19. Add 0.1 volume 3M sodium acetate to the final aqueous phase and fill the tube with 100% ethanol at -20°C.
20. Precipitate the chromosomal DNA for 3 - 5 minutes in a dry ice:ethanol bath, or for 15 - 30 minutes at -20°C or -80°C.
21. Recover the DNA by centrifugation. If even visible, the pellet will be clear, and may appear on the side of the tube.
22. Carefully decant the ethanol and sodium acetate supernatant.
23. Rinse the pellet once with -20°C 80% ethanol, and re-centrifuge.
24. Carefully decant the 80% ethanol wash.
25. Allow the pellet to dry at room temperature until no ethanol smell remains.
26. Resuspend the pellet in 50 µl to 200 µl of TE buffer or sterile dH2O.
† Proteinase K is a nonspecific serine protease. It is not inactivated by metal ions, chelating agents (e.g. EDTA), sulfhydryl reagents or by trypsin or chymotrypsin inhibitors. It is stable over a wide pH range (4 - 12.5), with optimal activity at pH 6.5 - 9.5. Activity can be stimulated by addition of denaturing agents (SDS and urea). The temperature optimum for the enzyme is 65°C; it is twelve times more active at 65°C than at 25°C. Rapid denaturation of the enzyme occurs at temperatures above 65°C.
‡ This and subsequent additions of TE serve to keep the sample size resonably high so you don't lose too much sample while removing the aqueous phase during each extraction step.
§ This usually requires 2 to 3 extractions.
Hope this helps!