Ed Beaty (edbeaty at uxa.cso.uiuc.edu) wrote:
: Hello. I've been using a spin-elution method to retrieve DNA fragments
: from agarose gels. Basically, I put the gel slice in an punctured
: eppendorf tube packed with glass wool and spin for 10 minutes
: at 10000 RPM in a microfuge. The liquid that passes through the glass wool
: is captured in a second eppendorf tube.
: The problem is that when I try to precipitate the DNA in the solution
: sqeezed from the gel slice, I get a large, insoluble pellet containing most
: of my DNA. I've tried the freeze-sqeeze technique and I get the same
: Phenol-Chloroform extraction of the eluate reduces the size of the pellet
: somewhat, but I still get an insoluble wad when I precipitate, even after
: several rounds of extraction. I've been using 0.1 volumes of 3 M sodium
: acetate (pH 5.2) and 2 volumes of cold absolute ethanol for my
: preciptiation, or 0.1 volumes of 3 M NaCl, and both give me insoluble
: pellets. It doesn't seem to matter whether the precipitation occurs at 4oC
: or room temperature. I've tried the various silica-chaotrope methods of
: DNA extraction talked about here, but I've never been really pleased with
: them (very low yeilds, degradation of the DNA). Any other ideas? I can
: get good precipitation of DNA with sodium acetate from reaction digests
: etc., but not after elution of DNA from an agarose gel. Help!
: Ed Beaty
another possible method to isolate fragments is the one of Jim Graham
(METHODS.FAQ Apr '94, 10.). All you have to do is run your fragments on
a LMP agarose gel, cut them out, melt the agarose, freeze it (I do it in
liquid nitrogen), thaw, centrifuge. The fragment is in the supernatant.
This methods works good in my hands. I usually precipitate by addition
of 2M ammonium acetate (endconcentration) and 2Vol. 96% EtOH and never
had problems to dissolve the DNA pellet.
Hope this helps
salger at zi.biologie.uni-muenchen.de