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 4°C)
• Cold 100% Ethanol (–20°C)
• Cold 70% Ethanol in sterile dH2O (–20°C)
• DNA sample
• Linear acrylamide
• 4°C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

1. Transfer DNA to a 500µl 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 1-3µl linear acrylamide, mix, and let stand in –20°C freezer for at least one hour.***
5. Centrifuge 14,000×g for 30 minutes at 4°C (to centrifuge we transferred reaction to a chilled 1.5µl tube).
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; 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.

* So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 120µl 100% EtOH, & 2µl 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 µg/ml... 2µl of a 5mg/ml stock seems to be the consensus elsewhere online... that would be 10µg in 188µl or ~50µg/ml, but that was fine.)

-- Main.LindseyWolf - 02 Dec 2011