undigestable genomic DNA

Jayakumar, R via methods%40net.bio.net (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
these reagents. 
Jayakumar
 

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu
[mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Ranadheer
Kumar Gupta
Sent: Thursday, October 18, 2007 2:56 AM
To: Tom Knight
Cc: methods from magpie.bio.indiana.edu
Subject: Re: undigestable genomic DNA

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.

regards
Ranadheer

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 
> Protocols.
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>

_______________________________________________
Methods mailing list
Methods from net.bio.net
http://www.bio.net/biomail/listinfo/methods


This email message may contain legally privileged and/or confidential information.  If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited.  If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you.



More information about the Methods mailing list