<noautolink> ---+ 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 H<sub>2</sub>O. 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) %RED% Be sure to store in a flammable safe freezer! %ENDCOLOR% * Cold 70% Ethanol in sterile dH<sub>2</sub>O (–20°C) %RED% Be sure to store in a flammable safe freezer! %ENDCOLOR% * DNA sample * Linear acrylamide (5 mg/ml) ([[http://products.invitrogen.com/ivgn/product/AM9520][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 dH<sub>2</sub>O to reach 200 µl. 1 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. 1 Add a volume of 100% ethanol equal to 2.5 times the *original* sample. Use ethanol that has been pre-chilled to –20°C. 1 *(Optional)* Add 2 µl linear acrylamide (10 µg) to the sample and mix. 1 Let stand in –20°C freezer for at least 30 minutes. In some cases longer in the freezer or overnight may improve recovery. 1 Centrifuge 14,000×g for 15 minutes at 4°C. 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. Pipet up and down until pellet dislodges from the side of the tube. 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 [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 H<sub>2</sub>O.
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Topic revision: r9 - 2021-06-11 - VictorLi