poly A selection
horton at molbio
Tue Aug 25 22:20:39 EST 1992
Greetings, denizens of cyberspace.
I have some theoretical/technical questions regarding selection of poly A+
RNA on oligo dT:
1) I've seen protocols for doing RNA hybridizations (e.g., for RNase
protection assays) in a guanidine solution. This supposedly has two effects;
it speeds up hybridization (however that works) and prevents any nasty RNases
from degrading the RNA prematurely. Could a guanidine-containing buffer be
used with an oligo dT cellulose column? (I get so nervous when my RNA has to
sit around at room temperature in water for an hour or so while I run these
1a) Can you keep azide (or something) on these columns all the time
to keep bugs from even thinking of growing on them? (And wouldn't they last
forever if nothing ate them?)
2) Is there a better method nowadays than oligo dT cellulose (e.g.,
magnetic beads, dT columns for HPLC, etc) that I am (hopefully) just unaware
of? (Dare I ask about kits people may like?)B-)) It seems like you could speed
up the process with a non-compressible matrix and a syringe (a poor man's
HPLC), but I'd hate to re-invent that wheel if somebody already sells 'em...
3) Or maybe I don't really need poly A+ RNA (I'm trying to label mRNA
for use as a probe on a panel of cloned genes. I've tried making hot cDNA from
total RNA using an oligo dT primer, but this gives a monstrous background).
Comments and suggestions would be greatly appreciated.
Bob Horton /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS || with nine women pregnant you get a baby a month"
1479 Gortner Ave. /||\ -Werner von Braun. Disclaimer:"Bob who?"
St. Paul, MN 55108 ^^ horton at molbio.cbs.umn.edu/(612) 624-3790
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