(r3) Barrick Lab :: ProtocolsEthanolPrecipitation

---+ 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

Barrick Lab > ProtocolList > ProtocolsEthanolPrecipitation

Contributors to this topic

LindseyWolf, GabrielSuarez, JeffreyBarrick, VictorLi, AlvaroRodriguez

Topic revision: r3 - 2011-12-19 - 20:38:50 - Main.LindseyWolf