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Help - RNA/DNA hybrid production

Robert Baker hall_lab at tikal.biosci.arizona.edu
Fri Jul 1 19:33:02 EST 1994


I'm trying to do an assay involving detecting RNase H activity in
re-natured proteins while in a polyacrylamide gel.  This involves
casting the gel with a radioactively labled RNA/DNA hybrid molecule,
renaturing the enzyme by washing away the SDS, and incubating the gel
so the enzyme can work on the substrate.  You the expose the gel and
look for light areas on the film.

Okay so far.  My question is: what's the best way to make the RNA/DNA
hybrid?

The paper I got this from is fairly old, and they mention using poly dT
(2000 bases long), and [alpha-32P]rATP and incubating with E. coli RNA
polymerase holoenzyme.  Will this work?  Does E. coli RNA polymerase
need a promoter to work, or not?

Any info would be very helpful.  Thanks in advance!

Robert Baker



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