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Transcription spots

Caroline A Breitenberger cbreiten at magnus.acs.ohio-state.edu
Fri Aug 27 15:18:15 EST 1993


Hello netters:  We have been having a recurring problem with in
vitro run-off transcription reactions in the presence of alpha-32P-
UTP from NEN, separated on 6% polyacrylamide-urea gels.  On the
autorad, we do see the expected product bands, but we also see
intense (and unpublishable!) black spots randomly scattered in
lanes in which transcription reactions were loaded.  Some of the
spots streak downward as if the label were electrophoresing out. 
Lanes containing markers (DNA labeled with gamma-32P-ATP) are not
spotty, however.  This leads me to suspect that something
particulate in the transcription reaction or the gel adsorbs the
32P label.
     When my student repeated the experiment in a different lab
with the same lysate and isotope, the autorad had NO spots.  He has
tried changing almost everything in the reaction and in the gel (in
our lab), to no avail.
     I called NEN to see if they had heard of this problem.  To
summarize the Techline's response, it was:  "Oh yes, we've heard of
that.  It's not our product, and it's not your assay, it's the way
the two are working together.  Bye."  (Huh?)
     Do any of you have similar problems/solutions?  (I will
summarize.)

ADVthanksANCE  (I saw someone else do this and I liked it!)
******************************************************************
Caroline Breitenberger
Ohio State University Department of Biochemistry
484 W. 12th Ave., Columbus, OH 43210
614-292-9473
caroline+ at osu.edu



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