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Newsgroups: bionet.molbio.methds-reagnts
From: Kyle Legate <lega...@hotmail.com>
Date: Thu, 18 Oct 2007 22:40:26 +0200
Local: Thurs, Oct 18 2007 4:40 pm
Subject: Re: undigestable genomic DNA
Ed Siefker wrote: I digest the tail under pretty much the same conditions, but for > 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. Southerns I do consecutive phenol and chloroform extractions (1 each) and precipitate using an equal volume of isopropanol. Air dry the pellet for a few minutes and dissolve in TE at 55C for as long as is necessary, with shaking if possible, or by raking the tube over an Eppi rack. The DNA will rarely fail to digest. You must Sign in before you can post messages.
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