RNAse protection assay
ks3f+ at andrew.cmu.edu
Mon Mar 13 17:03:34 EST 1995
I am trying to measure the kinetics of RNA:RNA pairing using an
RNAse protection assay. Briefly:
I initiate the pairing reaction (one 5' end-labelled RNA, one
unlabelled RNA). At time points, I take an aliquot of the pairing
reaction and digest for 15 sec with a large amount (10ug/ml) of RNAse A.
Reactions are stopped with phenol/chloroform, and the reaction is
separated on a sequencing gel.
The problem: Various time points totally disappear! For instance,
the 1 min. time point has 50% of the RNA protected, the 1.5 min. time
point has none of the RNA protected, and then the 2 min. time point has
75% of the RNA protected. The points that disappear vary from reaction
Dept. Biology, Carnegie-Mellon Univ.
Email ks3f@ andrew.cmu.edu
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