undigestable genomic DNA
(by akhan357 from sbcglobal.net)
Thu Oct 18 12:52:32 EST 2007
Dear Ranadheer, I had a tough time with strawberry leaf DNA during my Ph.D,
but finally was able to get good quality DNA for any downstream application.
methods bellow worked well for removal of sugars and subsequent isolation of
Manning, K. (1991) Isolation of nucleic acids from plants by differential
solvent precipitation. Anal. Biochem. 195: 45-50
or/and by using MasterPure Leaf DNA extraction kit (Epicenter Technologies)
you can also look at detailed method at
http://etd.lsu.edu/docs/available/etd-1027102-193854/ page 49.
"Ranadheer Kumar Gupta" <ranadheergupta from gmail.com> wrote in message
news:mailman.263.1192716952.23109.methods from net.bio.net...
> Hi all,
> I too got similar kind of problem, but it is with the plant leaf material.
> 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> wrote:
>> 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
>> Methods mailing list
>> Methods from net.bio.net
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