EtOH ppt of DNA, what's wrong with this strategy?

Chris LaRosa clarosa at biocomp.unl.edu
Fri Sep 8 09:16:33 EST 2000



"Dr. Hiranya S. Roychowdhury" wrote:
> 
> It most certainly will. Just make up a stock of 0.15M KOAc or NaOAC in
> ethanol (or 0.2M in 2-propanol) for the precipitation.
> The salt is not necessary if the DNA conc is very high. But usually it is
> not and the ionic conc. has to be high enough for the NA's to ppt. That
> seems to be about 0.3M for acetates and about 0.2M for chlorides.
> 
>I am looking at my mixture of Ethanol and sodium acetate and I see no precipitate and never have observed one.


For sequencing reaction ppt. 
I use a solution of 1 part 3M NaOAC pH 4.6 and 25 parts EtOH.  I just
examined the stock solution and there is no precipitate.  26 ul of this
is added to a 10 ul ABI BigDye reaction , set for 15 minutes and
centrifuged for 20 min @ 13000 rpm.  After removing the supernatent
carefully, the pellet is washed with 250 ul of 70 percent ethanol,
vortexed and spun down 5 min.   After carefully removing all liquid and
drying the sequencing reaction is ready for running.

this protocol worked far far better than other ppt protocols including
gel filtration for clean up.   

The lab adapted this protocol for precipitating pcr products as
templates for sequencing reactions with great sucess.  The ppt protocol
eliminated the need to use expensive silica based spin columns for pcr
reaction clean up. 
The following link gives the details of these protocols. 

http://lamarck.unl.edu/research/systematics/Orti/GO-lab/protocols/MasterProtocol.html






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