RNAse Protections

Jim Voeller voellerj at gunet.georgetown.edu
Wed Dec 13 14:22:36 EST 1995


In article <4ai760$joe at dub-news-svc-1.compuserve.com>, Tim Angelotti
<102055.606 at compuserve.com> wrote:

> I have a few practical questions about RNAse protection assays, for the 
> experts.  I am trying to see if a deletion mutant that I cloned by PCR is 
> real using a probe that spans the deletion.  I have found both a full 
> length band and a band corresponding to a cut at the site of the 
> deletion.  My question is how can I be sure that the cut at the deletion 
> site is due to the presence of an alternative transcript and not due to 
> secondary structure?  I am using Ambion's RPA II kit, hybridizing at 45 C 
> overnight.  The band is of the right size, but I am concerned that it may 
> be artifact.  Is this common, and would a higher temperature sort it out?

My suggestion at this point is to duplicate what you have done with a
wild-type non-mutated sample and run that sample alongside with your
previous deletion mutant. That may help you decide if you have artifact. I
am not familiar with the Ambion kit but my method uses a 85 C step before
placing at 45 C, this to melt secondary structure. How large is the
deletion?



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