Prep-A-Gene problems

Granger L. Ridout granger.ridout at murraystate.edu
Wed May 2 11:55:59 EST 2001


Hi!  I am trying to directly clean and gel clean PCR reactions of a 1.5
Kb bacterial community (sediment DNA) product using Bio-Rad's
Prep-A-Gene kit using anywhere from 5-20 uL of binding matrix, with
double binding matrix volume elution using either the kit's buffer or
deionized water (at 50 deg C) yet I keep losing my DNA, which gels
clearly show as there.  It has been suggested that this kit's binding
matrix, diatomaceous earth, I believe, has a tendency to stick to too
well to DNA and not give it up.  When I do get DNA it is only a few ug
out of 8 x 50 uL PCR reactions.  My other problem is that when I attempt
to do RE digestions on the "purified" DNA for RFLPs, the bands are there
but with a lot of background noise, which I have tried to destain
repeatedly, without success.  This includes both agarose (NuSieve) and
page with EtBr and silver staining.  Any ideas?  Thanks.

Granger




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