From Powers Lab UC Davis
1. Grow cells in 5 - 10 mls YPD
2. Spin cells down (8,000 rpm, 30s in microcentrifuge; 4,000 rpm, 10' in the Allegra tabletop); discard supernatant.
3. Wash cells. Add 1 ml ddH2O, resuspend, and spin again (same as above).
4. Resuspend cell pellet in 200 ul lysis buffer (see below).
5. Add 200 ul phenol/chloroform and 0.3 grams glass beads.
6. Eppendorf mix for 20' (tape tubes to mixer so that they don't spin around).
7. Spin 5' in a microfuge, 14,000 rpm.
8. Take aqueous phase. Add 3 ul of 10 mg/ml RnaseA. Incubate 10' at 37°C.
9. Add 200 ul TE.
10. Add 1X phenol/chloroform. Invert to mix. Microfuge, 5', 14,000 rpm. Take aqueous phase.
11. Add 1 ml 100% EtOH and NaCl (to a final concentration of 300 mM NaCl). Invert to mix. Place on ice 10'.
12. Spin in a microfuge, 10', 4°C cold room at 12,000 rpm.
13. Discard supernatant. (Be careful not to remove pellet!) Wash pellet in 1ml 70% EtOH. Spin 10', 4°C coldroom, 12,000 rpm.
14. Air dry approximately 5'.
15. Resuspend in 50 ul TE.
16. Use as is or make serial dilutions.
Lysis Buffer
10 mM Tris-HCl (pH 8.0)
100 mM NaCl
1 mM EDTA
2% Triton X-100
1% SDS
H2O
For 1ml:
1 M Tris-HCl (pH 8.0) 10 ul
3 M NaCl 33.3 ul
0.5 M EDTA 2 ul
20% Triton X-100 100 ul
10% SDS 100 ul
H2O 754.7 ul
TE = 10 mM Tris-Cl (pH 8.0) and
1 mM EDTA (pH 8.0)