undigestable genomic DNA

Ed Siefker via methods%40net.bio.net (by ebs15242 from creighton.edu)
Wed Oct 17 14:19:41 EST 2007


So I've been extracting mouse tail DNA for use in southerns for some 
time, and I keep running into DNA that won't digest. I use a pretty 
typical method.  I digest the tail piece with Proteinase K at 55C 
overnight in a buffer with 100mM NaCL and 1%SDS with some tris and EDTA 
as well. Then I extract with an equal volume of 
phenol/chloroform/isoamyl alcohol, followed by an extraction with just 
chloroform. Then I add 1/10 volume of 3M NaAc, 2 volumes of 100% EtOH 
and centrifuge.  Wash with 70% EtOH, dry, and resuspend in 10mM tris.

Frequently this DNA is very hard to resuspend, has a very high A230 
reading, and does not digest.  I gather this is polysaccharide 
contamination because the DNA is very globby and rather like snot. I 
have read that reprecipitation from 1M NaCL or 2.5M NH4Ac reduces 
polysaccharide contamination and it does make the DNA less globby and 
reduces the A230 reading, but I still cannot digest it.

Any suggestions would be most appreciated.



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