User:Anthony Salvagno/Notebook/Research/Phenol Extraction and Ethanol Precipitation

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I decided to write this protocol because I do this enough times. I have it memorized but who knows what will happen in the future and I may forget.

Phenol/Chloroform Extraction

You need a minimum sample volume of about 300uL. If you don't have this amount, bring the sample up to volume with either water or 1x TE or whatever other buffer you may have in solution.

  1. Pipette an equal amount of P/C (equal to your sample volume).
  2. Vortex it on high for about 30-60 sec. You want a good mixture.
    • This is called an immulsion.
  3. Spin at 13k rpm for 10 min.
  4. Pipette the top layer out and place in new test tube.
    • After the spin there will be 2 layers (maybe 3). The top is stuff you want and everything else is stuff you don't.
    • This is kinda stressful as after you pipette out most of the sample, the phenol will collapse upon the little remaining bit. You will still need to get to most of that little bit. Don't worry, some sample will be lost.

EtOH Precipitation

  1. After P/C extraction, add 1/10th volume of Ammonium Acetate to your new sample tube.
  2. Add about 1mL of EtOH to the tube.
    • If you have more than 500uL of sample split your sample into two tubes and add the EtOH to each tube.
  3. Place tube in freezer for at least one hour.
    • At this point you can leave the tube in the freezer for as long as you like, but one hour is the minimum time in case you need to get to work as soon as possible.
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