RNAse protection assay

[Pamela Norton]

In article <0098D58B.F170A75B at nefeli.imbb.forth.gr>,
skoufos at nefeli.imbb.forth.gr (Emmanuel Skoufos, Ph.D.) wrote:

> In article <cjNA4qK00iV847fqZq at andrew.cmu.edu>, Katherine Schaefer <ks3f+ at andrew.cmu.edu> writes:
> >

> >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. 
> 
> It seems that you are using way too much RNAse A.  Try to find the optimum
> concentration of RNAse for your experiment.  The concentration that I use
> when I do RNAse protection experiments (DNA:RNA hybrids,though) is 25 ng/ml.

   Some stuff edited away:

   Optimization of RNAse concentration to use much lower levels may require
lengthier incubation times. However, it is possible to use the 10 ug/ml
that is standard in many protocols. A proteinase K digestion step should
follow the RNAse step, then phenol/chloroform. The proteinase K step can be
omitted with the lower RNAse concentrations recommended by Emmanuel. 

   Hope this helps,

								Pam Norton