undigestable genomic DNA
(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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