Spin-Elution and ppt. of DNA

Ed Beaty edbeaty at uxa.cso.uiuc.edu
Wed Jul 6 16:42:59 EST 1994


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
problem.  

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!


Thanks,
Ed Beaty
edbeaty at uxa.cso.uiuc.edu



More information about the Methods mailing list