(r6) ProtocolsEthanolPrecipitation < Lab

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---+ 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>. 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)
   * 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 Add 2 µl linear acrylamide (10 µg) to the sample, mix, and 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&times;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*: When precipitating single-stranded nucleic acid samples it may improve recovery to resuspend in 0.001% SDS to prevent sticking to the wall of the tube.
Barrick Lab > ProtocolList > ProtocolsEthanolPrecipitation
Topic revision: r6 - 2013-05-25 - 02:40:48 - Main.GabrielSuarez