1. Pellet 6 or 7 ml ON culture.
2. Aspirate off supernatant completely.
3. Add 100 µl solution I and resuspend pellet.
4. Incubate @ RT 5 min.
5. Add 200 µl solution II, mix gently by inverting the tube.
6. Incubate on ice 5 min.
7. Add 150 µl solution III, mix gently by inverting the tube.
8. Incubate on ice 5-10min.
9. Centrifuge 5 min @ 14 K rpm.
10. Remove 400 µl supernatant carefully to new tube.
11. Add 800 µl cold 95% EtOH, invert the tube several times.
12. Incubate @ RT 2 min.
13. Remove supernatant and stand the tube upside down.
14. Resuspend in 50 µl of 0.1M NH₄OAc.
15. Add 150 µl cold 95% EtOH.
16. Incubate 2 min @ RT.
17. Centrifuge 5 in @ 14 K rpm / RT.
18. Remove EtOH completely.
19. Resuspend in 100 µl dH₂O.
20. Add 1 µl RNase (1mg/ml) incubate 30 min @ 37C.
21. Run 10 µl on the agarose gel to check for purity and concetration.
22. Add 110 µl H₂O, 4 µl 5M NaCl & 200 µl phenol.
23. Vortex, spin and remove upper phase to a new tube.
24. Add 200 µl chl./isoamylOH 24:1 vortex & remove upper phase
to a new tube (this step do twice in a row).
25. Add 1/10 vol 3M KAc & 3 vol 100% EtOH & precipitate @ -80C
for 30min. Spin @ 14k rpm for 5min.
26. Wash with 500 µl 70% EtOH, spin.
27. Pour off alcohol, dry & resuspend in H₂O