undigestable genomic DNA
(by R.Jayakumar from roswellpark.org)
Thu Oct 18 11:24:56 EST 2007
Plant DNA is tough and depends on the type of plants. Monocots are easy
and much cleaner, but dicots are just so difficult, especially if the
plant is an aromatic or an oil seed variety.
Could try usin N-Lauryl sarcosine to clear up the polysaccharides and
PVP for clearing up the polyphenolics. Look for a protocol that uses
From: methods-bounces from oat.bio.indiana.edu
[mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Ranadheer
Sent: Thursday, October 18, 2007 2:56 AM
To: Tom Knight
Cc: methods from magpie.bio.indiana.edu
Subject: Re: undigestable genomic DNA
I too got similar kind of problem, but it is with the plant leaf
Can it be reduced by Urea purification?
If any of you have some tips, please help me.
On 17 Oct 2007 17:08:00 -0400, Tom Knight <tk from shaggy.csail.mit.edu>
> 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
> > 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
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