Summers, Zarath 1-2
DNA Extraction Protocol for Methanogens
1.
2.
3.
4.
5.
6.
7.
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 ul 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 ul of chloroform
16. Vortex for 15 seconds to mix.
17. Centrifuge at 16,000xg for 15 minutes at 4oC.
18. Transfer 600 ul of aqueous phase to a new tube to use for RNA extraction. Be
careful to avoid the interphase.
19. The phenol phase and interphase can be stored overnight at 4oC.
20. Remove remaining aqueous phase overlying the interphase. (Removal of the
aqueous phase is critical for the quality of the isolated DNA)
21. Add 300 ul of 100% ethanol
22. Mix by inversion.
23. Incubate at room temperature for 2-3 min.
24. Centrifuge at no more than 2,000x g for 5 minutes at 4oC.
25. Remove the phenol-ethanol supernatant.**
26. Add 1 ml of 0.1 M sodium citrate in 10% ethanol.
27. Incubate at room temperature for 30 min, mixing periodically.
28. Centrifuge at 2,000x g for 5 minutes at 4oC.
29. Remove supernatant.
30. Repeat steps 26-29 one time. (An additional 0.1M sodium citrate in 10% ethanol
wash step is required for large pellets containing >200 ug DNA or large amounts
of non-DNA material.)
31. Re-suspend pellet in 1.5 ml of 75% ethanol
32. Incubate at room temperature for 10-20 minutes, mixing periodically.
a. Samples suspended in 75% ethanol can be stored at 4oC for months.
33. Centrifuge at 2,000x g for 5 minutes at 4oC.
34. Air-dry DNA in an open tube for 10-15 minutes.
35. Add 300 ul of 8 mM NaOH to DNA
36. The pH of the solution should be adjusted to ~9 with TE or HEPES once the DNA
is in solution.
Summers, Zarath 2-2
37. The solution may contain insoluble gel-like material (membrane fragments, ect.)
a. Remove this material by centrifugation at 12,000x g for 10 minutes at 4oC
b. Transfer the supernatant containing the DNA to a new tube.
38. DNA solubilized in 8 mM NaOH can be stored overnight at 4oC.
39. For prolonged storage samples should be adjusted with HEPES to pH 7-8 and
supplemented with 1mM EDTA.
pH adjustment of 1ml 8mM NaOH using 0.1 M HEPES
Final pH
0.1M HEPES (ul)
8.4
86
8.2
93
8.0
101
7.8
117
7.5
159
** Phenol ethanol supernatant can be saved for protein isolation.
Adapted from Invitrogen Form No. 18057N by Z.S. 9/21/2006