Summer, Zarath 1-1
RNA Extraction Protocol for Methanogens
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In 50 ml Falcon tubes pellet ~100 ml culture.
Re-suspend cell pellet in 1ml of water
Transfer mix into 1.5 ml blue RNase-free tube
Re-pellet cells
Remove supernatant.
Dip tip of blue 1.5ml tube into liquid nitrogen to freeze cell pellet
Grind cell pellet with blue pestle for 3 minutes, while continuing to dip the tip of the
tube into liquid nitrogen to keep pellet frozen.
8. Add 1ml TRIzol to the frozen cell powder.
9. Pipette up and down to thaw powder.
10. Transfer mix into a 2ml O-ring tube containing 100 l each of 0.1 mm and 0.5 mm
glass beads.
11. Bead-beat for 3 minutes
12. Let stand for 5 minutes rest
13. Bead-beat for 3 minutes
14. Incubate at room temperature for 5 minutes.
15. Add 200 l of chloroform
16. Vortex for 15 seconds to mix.
17. Centrifuge at 16,000xg for 15 minutes at 4C.
18. Transfer 600 l of aqueous phase to a new tube. Be careful to avoid the interphase.**
19. Add 600 l 2-propanol
20. Mix
21. Incubate on ice for 15 minutes.
22. Centrifuge at 16,000xg for 15 minutes at 4C.
23. Remove the supernatant
24. Wash the pellet with 750 l 70% ethanol
25. Air dry.
26. Re-suspend pellet in 50-100 l of RNase-free water.
** The interphase and phenol phase can be kept for DNA extraction.
Adapted from: D.E. Culley et al. Journal of Microbiological Methods 67 (2006) 36-43 by
Z.S. 9/21/2006