Leonard N. Bloksberg
bloksber at pilot.msu.edu
Mon Oct 25 16:50:00 EST 1993
In Article <9310251625.AA06763 at ccu.umanitoba.ca> "ramacha at CC.UManitoba.CA" says:
> Hello Netters,
> I have a question....
> What is the significant differences between precipitating DNA with
> isopropanol at RT versus salt and ethanol in the cold.
> I accidentally precipitated some DNA (Already in a high salt buffer) with
> Ammonium acetate and 95% EtOH instead of using isopropanol according to
> the protocol. Do you think there will be a dofference in the yield elc.,.
> Any comments / suggestions are welcome.
> Thank you for your help
> Karthikeyan R.
> Dept. of Microbiology Ph(Off) 204 474-9227
> Univ. of Manitoba Ph(Res) (204) 275-7812
> Winnipeg, Manitoba Fax. (204) 275-7615
> Canada R3T 2N2 e-mail ramacha at ccu.umanitoba.ca
I don't think you will have a problem. Increased salt will not inhibit
precipitation of nucleic acid, it just leaves more junk to wash out in the
70% EtOH wash. If you think you have too much salt, just do 2 70% EtOH
washes (minimum of 10min each) if it makes you feel better. As for EtOH
vs. propanol, the difference is nominal. Ethanol (as I recall) precipitates
more efficiently (especially for small fragments) but propanol requires less
volume. The thing I always think about in differential precipitation as a
purification step is "what am I trying to optimize?" More alcohol (2.5 vs.
2 volumes of ethanol) or lower temperature, will increase efficiency of
precipitation. For clean, dilute, small DNA samples, high efficiency is
very important, but for dirty preps of concentrated, large DNA (alk. lysys
plasmid minipreps) it can be detrimentatl. Here, lower efficiency means
better separation from contaminants. There was an old review in Methods in
Enzymology that I read in grad school that went over all these parameters.
The bottom line is that alcohol precipation of DNA is VERY robust, and will
probably work fine under a vast array of conditions.
. Leonard N. Bloksberg
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