Good news - we're open for limited services in Wembley. Ujima House is now actively under refurbishment and we'd love your help in making the space the best it can be.

Please pay attention to the main LHS mailing list or pop into our #london-hack-space IRC channel and say hello.

During this interim period donations and continued membership are greatly appreciated while we transition to our new space.

DNA extraction and precipitation with ethanol / isopropanol

From London Hackspace Wiki
Jump to: navigation, 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.