Difference between revisions of "DNA extraction and precipitation with ethanol / isopropanol"
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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. | ||
Revision as of 11:29, 28 October 2012
- 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.