---+ 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. 1 Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations. 1 Add at least two volumes of cold 100% ethanol. 1 Add 1-3ul linear acrylamide, mix, and let stand in -20°C freezer for at least one hour.*** 1 Centrifuge 14,000xg for 30 minutes at 4°C (to centrifuge we transferred reaction to a chilled 1.5ul tube). 1 Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200ul pipet. 1 Add 200ul of cold 70% ethanol; centrifuge for 10 minutes at 4°C. 1 Remove supernatant with a 200ul pipet; evaporate remaining ethanol in a 37°C water bath or heat block. 1 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
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Topic revision: r3 - 2011-12-19 - LindseyWolf