undigestable genomic DNA

Tom Knight via methods%40net.bio.net (by tk from shaggy.csail.mit.edu)
Wed Oct 17 16:08:00 EST 2007


Ed Siefker <ebs15242 from creighton.edu> writes:

> 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.

You might try a second round of phenol/chloroform, or (if it really is
polysaccharides) isolation using CTAB instead of SDS.  See Current
Protocols.


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