Purifying DNA from gel slices
Peter S. Cooper
cooper3 at niehs.nih.gov
Fri Nov 8 12:34:33 EST 1996
David Bird wrote:
> Alison Reavley wrote:
> > Hi, I'm looking for a method of purifying plasmid DNA from gel slices
> > which does not involve phenol or any increase in temperature. At the
> > moment I am trying electroelution but find that I am precipitating agarose
> > as well as DNA after the elution. I need to get rid of this agarose again
> > without phenol or heating. I have also tried using Quiagen Qiaquick Gel
> > Extraction columns but have been frustrated by the small yield and I am
> > now using concentrations of DNA above those recommended in the protocol.
> > Any suggestions?
> > Alison Reavley
> > MRC Radiation and Genome Stability Unit,
> > Harwell,
> > Didcot UK.
> Many people in my department have used Glassmilk (pulverized glass
> beads) with success. This preferentially binds DNA to glass beads, and
> the DNA is later eluted with a salt buffer, after washing with EtOH. It
> is supposed to give 80% recovery; I have never been so successful, but
> many prefer this method.
The GeneClean Kit from BIO101 which uses powdered glass (Glassmilk) has
worked quite well in my hands. I don't know about 80% recovery but it
the yield is much higher than with LMP agarose phenol extraction.
HOWEVER this method does not work well for smaller fragments < 700 bp.
You might try a freeze-squeeze type protocol. You need to prepare some
kind of spin filter though. You basically cut out the gel slice
containing the band of interest. Then freeze it overnight at -70. Thaw
it out and spin the heck out of it through the spin filter.
Peter S. Cooper, Peptide Neurochemistry, Mail Stop C3-04
National Institute of Environmental Health Sciences
P.O. Box 12233
Research Triangle Park, NC 27709
Phone: (919) 541-3238
FAX: (919) 541-0626
email: cooper3 at niehs.nih.gov
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