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 H₂O. 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) Optional addition when DNA concentration is low, binds and helps to create visible precipitate, not necessary with higher DNA concentrations, such as with phage genomic DNA extraction
• 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. (Optional) Add 2 µl linear acrylamide (10 µg) to the sample and mix.
5. Let stand in –20°C freezer for at least 30 minutes. In some cases longer in the freezer or overnight may improve recovery.
6. Centrifuge 14,000×g for 15 minutes at 4°C.
7. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
8. 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.
9. 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.
10. 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: To prevent single-stranded nucleic acids from sticking to the tube's side walls one can resuspend in 0.001% SDS + TE or 0.001% SDS + Nanopure H₂O.