RNAse protection assay

Katherine Schaefer 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
to reaction. 
    Any thoughts?
       Katherine Schaefer
       Dept. Biology, Carnegie-Mellon Univ.
       Email ks3f@ andrew.cmu.edu 

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