DNA extraction and precipitation with ethanol / isopropanol
From London Hackspace Wiki
Sample collection
- Collect a small amount of cells by gently scraping the interior of your cheek.
- Transfer the collected cells to a 1.5 ml tube.
Novel experimental procedure for DNA extraction (28/10/12)
(This has not yet been tested)
- Add to the cell sample 200 µl of Lysis Buffer (10 mM Tris-HC1 (pH 8.0), 1 mM EDTA, 1% Triton X-100).
- Incubate solution at 94˚C for at least half an hour (better 1h).
- Spin in microcentrifuge at 13,000 rpm for 10 min.
- Collect supernatant in a novel tube and discard pellet. Procede with proteinase K treatment and ethanol precipitation.
Proteinase K treatment and ethanol precipitation
- Get DNA sample from previous extraction.
- Add 1 part proteinase K (1mg/ml stock solution) to 3 parts sample. Incubate at 50˚C for 1 hour and then heat-inactivate proteinase K at 94˚C for 10 min.
- Add 1/10th volume 3M sodium acetate pH 5.5. Mix by inverting tube.
- Add 2-3 volumes of cold 95-100% ethanol. Mix by inverting tube.
- Spin in microcentrifuge at 13,000 rpm (if possible at 4˚C) for 10-15 min.
- Aspirate supernatant carefully not to disturb pellet.
- Add 500 µl 70% ethanol (stored at -20˚C).
- Spin in microcentrifuge at 13,000 rpm for 2 min.
- Aspirate supernatant. Dry pellet at room temperature for 10 min (can be heated gently).
- Resuspend in water or TE buffer. TE buffer is generally preferred, because the DNA would be more stable. If the sample does not redissolve it can be heated at 50-60˚C for half an hour to help it.
