Ethanol Precipitation

Precipitating DNA/RNA from solution to remove salts and small nucleic acid fragments.

Materials

  • 3M Sodium Acetate, pH 5.2 (store at 4C)
    • Make by dissolving 408.3 g sodium acetate trihydrate in ~800 ml H2. Titrating pH with glacial acetic acid in a hood (being careful of the fumes) to pH 5.2. And then adding water to reach final volume of 1 L.
  • Cold 100% Ethanol (20C) Be sure to store in a flammable safe freezer!
  • Cold 70% Ethanol in sterile dH2O (20C) Be sure to store in a flammable safe freezer!
  • DNA sample
  • Linear acrylamide (5 mg/ml) (Ambion Catalog #AM9520) (function of polyacrylamide: carrier that increases efficiency of DNA precipitation)
  • 4C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

  1. Transfer DNA to a 1.7 ml Eppendorf tube. If your sample volume is < 200 l, it can be helpful to add dH2O to reach 200 l.
  2. Add a volume of Sodium Acetate equal to one tenth the sample volume. This provides the high ion concentration and right pH for the DNA to precipitate.
  3. Add a volume of 100% ethanol equal to 2.5 times the original sample. Use ethanol that has been pre-chilled to 20C.
  4. Add 2 l linear acrylamide (10 g) to the sample, mix, and let stand in 20C freezer for at least 30 minutes. In some cases longer in the freezer or overnight may improve recovery.
  5. Centrifuge 14,000×g for 15 minutes at 4C.
  6. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200l pipet.
  7. Add 200l of cold 70% ethanol. Pipet up and down until pellet dislodges from the side of the tube. Centrifuge for 10 minutes at 4C.
  8. Remove supernatant with a 200l pipet. Evaporate residual ethanol using a speed-vac or by opening the tube and leaving at room temperature or in a 37C heat block.
  9. Resuspend pellet in water or a new buffer of choice to an appropriate concentration [2].

* So for a 60l ligation reaction: 60l DNA, 6l Sodium acetate, 150l 100% EtOH, and 2 l acrylamide.

Variation: When precipitating single-stranded nucleic acid samples it may improve recovery to resuspend in 0.001% SDS to prevent sticking to the wall of the tube.

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Topic revision: r6 - 25 May 2013 - 02:40:48 - Main.GabrielSuarez
 
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