oligo hybridization

John Cooper Picard at nih.gov
Fri Nov 4 17:15:47 EST 1994


In article <199410271410.HAA01100 at net.bio.net> fishgen at vt.edu (Bruce  J. Turner) writes:
>From: fishgen at vt.edu (Bruce  J. Turner)
>Subject: oligo hybridization
>Date: 27 Oct 1994 07:11:00 -0700
>In article date 10/27/94, Limin Li requests help with oligonucleotide
>hybridization.  Although we have successfully hybridizewd oligos to
>blotting membranes, we consistently obtain much better results by
>hybridizing them directly to dried agarose gels.  Background is reduced,
>and hybridization efficiency seems much higher.  This is an old technique
>(originally from Cliff Brunk's lab at UCLA, I believe), but, so far as
>we're concerned, it's a good one as well (see PNAS 89:10643, 1992 and
>87:5653, 1990, for examples from our lab).  Essentially, the dried gel is
>treated as if it were a membrane, denatured and hybridized, etc.  We have
>gotten up to 10 repeated hybridizations with the same dried gel and
>routinely do 3 or 4.  The technique works well for oligomers up to about 40
>bases and best in the 20-30 range.  It is apparently limited to P-32
>labeled oligos (the reagents involved in most nonradioactive detection
>systems don't penetrate the dried agarose very well) but otherwise is a
>breeze.
>
>
John Cooper, MD
National Inst. of Health
Picard at helix.nih.gov



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