EtOH precipitation temp

jim hartley jhartley at access2.digex.net
Mon Jan 22 10:08:40 EST 1996


Many years ago now we investigated the effect of temperature and time on 
recoveries of 32P DNA by ethanol precipitation.  The bottom line was that 
if you have an hour to spend, you are far better off centrifuging for an 
hour and not cooling the precipitation reaction at all.  So for a decade 
I have been adding three volumes of 0.1 M sodium acetate in ethanol (this 
stock is stable indefinitely at room temp) to my DNA solutions 
and centrifuging immediately at room temperature.  Unless there is some 
chemical or enzymatic reaction that happens in 75% ethanol, there is no 
reason to do this cold.  Cooling may actually hurt recovery, if the 
viscosity of the solution is increased.  
(The precipitate has to travel to the wall of the tube to be recovered.)
Five to ten minutes in a microfuge and on to the next step.  Incubation 
in dry ice/ethanol prior to centrifuging decreased recovery, presumably 
because of increased viscosity.  Incubation overnight at -20 helped 
recovery a little, but this makes sense only for really low 
concentrations of valuable DNA (in our case, 10 ng DNA in a 1 ml ethanol 
precipitation).



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