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DNA extraction and precipitation with ethanol / isopropanol: Difference between revisions
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DNA extraction and precipitation with ethanol / isopropanol (view source)
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* 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. | ||
* | * 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. | ||
* Resuspend in | * 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. | |||
