DNA extraction of aquatic bacteria communities
DO NOT PUBLISH WITHOUT PERMISSION OF AUTHORS
Feng Chen and Jinjun Kan
1. Collect 500ml samples and filtrate it through 0.22 µm pore-size polycarbonate membrane filter (47mm in diameter, Millipore). Note: change the filter when the flow rate is slow.
2. Place the filter(s) into a Whirl-Pak bag and store the bag at –20 C (only if you are not going to extract the DNA right after this).
3. Thaw the filters in ice and add 2ml pre-lysis buffer (Tris-HCl, 0.1M; EDTA, 0.1M; sucrose, 0.8M), 10 µl lysozyme (200µg/µl), and incubate the sample at 37°C for 30 min.
4. Add 10 µl proteinase K (20 mg/ml) and SDS (final concentration: 1%), respectively, and incubate the sample at 37°C overnight.
5. Divide the sample into two microcentrifuge tubes, 1ml for each tube.
6. Set the incubator temperature at 65°C. Add 100 µl CTAB (10%) +NaCl (1.4M) to each tube, and incubate both tubes at 65°C for 30 min.
7. Add equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) to each tube, mix and centrifuge both tubes at 13,000 rpm for 15 min.
8. Take supernatent, and add equal volume of chloroform-isoamyl alcohol (24:1) to the supernatent, centrifuged at 13,000 rpm for 5 min.
9. Repeat 5-6 if necessary.
10. Take supernanant, add equal volume of isopropanol or 2 volume of cold ethanol (-20°C) to the supernatent, place
the tubes at -80°C for 20 min or –20 °C for 2hr.
11. Centrifuge both tubes at 13,000 rpm for 15 min, wash DNA pellet with 70% ethanol twice.
12. Dry DNA pellet with Speedvac for ~10 min.
13. Add 100µl double-distilled water to one tube and keep the other tube dry as a backup.
