Ethanol Precipitation

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

Materials

• 3M Sodium Acetate, pH 5.2 (store at 4°C)
  • 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 (–20°C) Be sure to store in a flammable safe freezer!
• Cold 70% Ethanol in sterile dH₂O (–20°C) 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)
• 4°C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

1. Transfer DNA to a 1.7 ml Eppendorf tube. If your sample volume is < 200 µl, it can be helpful to add dH₂O to reach 200 µl.
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 –20°C.
4. Add 2 µl linear acrylamide (10 µg) to the sample, mix, and let stand in –20°C 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 4°C.
6. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
7. Add 200µl of cold 70% ethanol. Pipet up and down until pellet dislodges from the side of the tube. Centrifuge for 10 minutes at 4°C.
8. Remove supernatant with a 200µl pipet. Evaporate residual ethanol using a speed-vac or by opening the tube and leaving at room temperature or in a 37°C heat block.
9. Resuspend pellet in water or a new buffer of choice to an appropriate concentration [2].

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