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EthanolPrecipitationDNA.md

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Ethanol Precipitation of DNA

This protocol is generally used in the lab to concentrate and purify DNA that has already been extracted. It is also commonly used in the lab after performing a DNA extraction using DNAzol, as the buffer used for that is not useful for most downstream applications (like genome resequencing).

This version of the protocol has been adapted from that in Sambrook and Russell (Molecular Cloning: A laboratory manual, third edition, 2001 CSHL publishing), in appendix A8.12-A8.14. Lots of useful advice in that section if you need to do any troubleshooting.

Beware that the DNA pellet is often very small (and barely visible). Be very careful during aspiration to avoid disturbing or losing the pellet.

  1. Add 1/10th volume (relative to volume of DNA) of 3M sodium acetate (pH 5.2-5.5), such that the final concentration of sodium acetate is 0.3M. (example if your DNA is currently in 90microL of some buffer you would add 10microL of 3M sodium acetate). Mix by inverting tube (not by pipetting).

  2. Add 2 volumes of 100% EtOH (ice cold). Mix by inverting tube. If the amount of DNA is small (from 1-20 flies) I recommend incubating on ice for 20-30 minutes.

  3. Spin in microcentrifuge at 14000 rpm at 0-2 degrees Celsius for 15 minutes (25 minutes for low concentrations or small amounts of DNA). (note this time can be extended to an hour or more for very minute amounts of DNA. Also consider adding MgCl2 to a final concentration of 0.01M in such cases).

  4. Aspirate all supernatant carefully and do not disturb pellet.

  5. Add 500 microL of 70% ethanol (stored at -20 degrees Celsius).

  6. Spin in microcentrifuge at 14000 rpm at 4 degrees Celsius for 2 minutes. (note: if there is a concern of additional contaminants step 4 and 5 can be repeated).

  7. Aspirate supernatant. Dry pellet at room temperature for ten minutes (until last traces of fluid have evaporated).

  8. Resuspend DNA in water or TE buffer (pH 7.6-8.0). TE is generally preferred as the DNA is more stable. Mak sure to wash walls of tube with the TE buffer.

For resequencing purposes we generally want final DNA concentrations of 100 nanograms/microL. Assume that you will lose between 25-50% of DNA during the ethanol precipitation.