Restriction enzyme problems

Stacy Ferguson stacyef at stacyef.net
Sat Dec 11 01:59:41 EST 2004


In article <51e45ae.0412030358.7c6155a0 at posting.google.com>,
 thomaskkristensen at hotmail.com (Thomas Kristensen) wrote:

> Dear All.
> 
> I have a problem cutting DNA extracted from fish tissue that has been
> preserved in ethanol 96% for ~1 wk.
> I am extracting with phenol and then phenol:chloroform (1:1) pH 8.0
> and then precipitating with NaAc 3M and isopropanol and washing twice
> with EtOH before dissolving the pellet in TE.
> Usually this procedure does not cause any problems to the following
> digestion but with ethanol preserved samples BamHI and BglII did not
> work...
> 
> Is ethanol preservation generally a problem when extracting DNA for
> digestion?

No. In my hands, new genomic DNA preps that won't cut will usually 
digest fine after overnight dialysis against TE (I usually go with 2 
liters). I throw it on a stir plate, take the DNA out of the dialysis 
bag the next morning, re-check the OD260/280 and do another test digest. 
This solves my problem at least 9 out of 10 times whether I made the DNA 
myself or got it from someone else. 

Stacy



More information about the Methods mailing list