Anonymous

DNA extraction and precipitation with ethanol / isopropanol: Difference between revisions

From London Hackspace Wiki
no edit summary
No edit summary
No edit summary
Line 1: Line 1:
* Get DNA
* Get DNA sample from previous extraction.
* Add 1 part proteinase K to 3 parts sample
* 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 ethanol
* Add 1/10th volume 3M sodium acetate pH 5.5. Mix by inverting tube.
* Centrifuge and pour away ethanol
* Add 2-3 volumes of cold 95-100% ethanol. Mix by inverting tube.
* Wash with 70% ethanol
* Spin in microcentrifuge at 13,000 rpm (if possible at 4˚C) for 10-15 min.
* Air dry for ∼10 mins. Can be heated gently.
* Aspirate supernatant carefully not to disturb pellet.
* Resuspend in dH20 or TE. If the sample isn't dissolving it can be heated at 50-60C to help it.
* 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.
8

edits