(r4) Barrick Lab :: ProtocolsEthanolPrecipitation

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---+ 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&times;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

Barrick Lab > ProtocolList > ProtocolsEthanolPrecipitation

Contributors to this topic

LindseyWolf, JeffreyBarrick, GabrielSuarez, VictorLi, AlvaroRodriguez

Topic revision: r4 - 2011-12-19 - 21:25:29 - Main.JeffreyBarrick