Ethanol Precipitation

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

Materials

  • 3M Sodium Acetate buffer, pH 5.2 (store at 4C)
  • Cold 100% Ethanol (20C)
  • Cold 70% Ethanol in sterile dH2O (20C)
  • DNA sample
  • Linear acrylamide
  • 4C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

  1. Transfer DNA to a 500l tube.
  2. Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.
  3. Add at least two volumes of cold 100% ethanol.
  4. Add 1-3l linear acrylamide, mix, and let stand in 20C freezer for at least one hour.***
  5. Centrifuge 14,000×g for 30 minutes at 4C (to centrifuge we transferred reaction to a chilled 1.5l tube).
  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; 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.

* So for a 60l ligation reaction: 60l DNA, 6l Sodium acetate, 120l 100% EtOH, & 2l acrylamide/

(there's an interesting typo in the Ambion protocol for this... the acrylamide stock is 5mg/ml and the protocol says you want a final concentration of 10-20 g/ml linear acrylamide... I assume they meant g/ml... 2l of a 5mg/ml stock seems to be the consensus elsewhere online... that would be 10g in 188l or ~50g/ml, but that was fine.)

-- Main.LindseyWolf - 02 Dec 2011

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Topic revision: r4 - 19 Dec 2011 - 21:25:29 - Main.JeffreyBarrick
 
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