<noautolink> ---+ 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. 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 1-3µl linear acrylamide, mix, and let stand in –20°C freezer for at least one hour.*** 1 Centrifuge 14,000×g for 30 minutes at 4°C (to centrifuge we transferred reaction to a chilled 1.5µl tube). 1 Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet. 1 Add 200µl of cold 70% ethanol; centrifuge for 10 minutes at 4°C. 1 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. 1 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
This topic: Lab
>
WebHome
>
ProtocolList
>
ProtocolsEthanolPrecipitation
Topic revision: r4 - 2011-12-19 - JeffreyBarrick