Ethanol_precipitation

Precipitating out salts from DNA samples

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 500ul 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 1-3ul linear acrylamide, mix, and let stand in -20°C freezer for at least one hour.***
5. Centrifuge 14,000xg for 30 minutes at 4°C (to centrifuge we transferred reaction to a chilled 1.5ul tube).
6. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200ul pipet.
7. Add 200ul of cold 70% ethanol; centrifuge for 10 minutes at 4°C.
8. Remove supernatant with a 200ul pipet; evaporate remaining ethanol in a 37°C water bath or heat block.
9. Resuspend pellet in 50ul of water.

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

-- Main.LindseyWolf - 02 Dec 2011