DNA extraction and precipitation with ethanol / isopropanol

From London Hackspace Wiki
Jump to navigation Jump to search

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.