1. Pellet 1 or 1.5 ml ON culture.
2. Aspirate off supernatant completely.
3. Add 100 µl solution I and resuspend pellet.
4. Add 200 µl solution II, mix gently by inverting the tube.
5. Incubate on ice 5 min.
6. Add 150 µl solution III, mix gently by inverting the tube.
7. Incubate on ice 5-10min.
8. Centrifuge 5 min @ 14 K rpm.
9. Remove 400 µl supernatant carefully to new tube.
10. Add 800 µl cold 95% EtOH, invert the tube several times.
11. Incubate @ RT 2 min.
12. Centrifuge 5 min @ 14 K rpm / RT.
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 50 µl dH₂O.