genomic DNA purification questions

nishir at ohsu.edu nishir at ohsu.edu
Wed Aug 17 12:08:03 EST 1994


In article <9408161553.AA13566 at post.its.mcw.edu> FRED at bmt.mcw.edu ("Frederick
Garbrecht") writes:
>A neophyte (me) needs help.  When I am purifying genomic DNA from 
>human lymphoid cells, I have a lot of trouble in the 
>phenol-chloroform extractions - when I pipette off the upper aqueous 
>phase, the DNA strands always seem to be attached to the junk at the 
>interphase, so consequently I pull up a lot of the crap in the 
>interphase and can't seem to get rid of it.  Any suggestions?  Do 
>people just take off half of the aqueous phase and sacrifice the rest 
>to get a clean preparation?  Thanks much --
>
>Frederick C. Garbrecht
>fred at bmt.mcw.edu
>fgarbrec at post.its.mcw.edu
>Bone Marrow Transplant Program
>Medical College of Wisconsin
>phone 414 257 5053
>fax   414 257 7994
>

You can reduce the amount of crap that you pull up by using a pipet tip that
has a large diameter (eg., when we do phenol: CHCl3 extractions in microfuge
tubes we remove the aqueous phase with wide mouth pipet tips or cut off the end
of the yellow pipet tip).  If the DNA is precious and you don't want to give
the stuff you lose by being conservative and taking 1/2 to 2/3 of he aqueous
phase, then you can back extract the phenol: CHCl3.  Take the stuff you leave
behind and add an equal volume of TE or water and extract again; pool with your
other stuff.  I don't bother to do this if I'm extracting genomic DNA because
you usually get so much; I do back extract if I'm cleaning up lambda DNA.

Rae Nishi
OHSU
Portland OR 




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