undigestable genomic DNA

Kyle Legate via methods%40net.bio.net (by legatek from hotmail.com)
Thu Oct 18 15:40:26 EST 2007


Ed Siefker wrote:
> 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.
> 
I digest the tail under pretty much the same conditions, but for 
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.


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