RNase inhibitor does not work ???

Richard M. Rohan rrohan at umabnet.ab.umd.edu
Thu Feb 9 20:53:51 EST 1995


> I tested 45U of RNase inhibitor 
> from Ambion in a 10 ul reaction volume with 2 ug of good RNA preparation and 
> 50 ng of RNase A. The buffer I used contained 50 mM Tris HCl, pH 8.2, 10 mM 
> MgCl2, and 10 mM DTT. The reaction was incubated at 30C for 90 minutes. 


One unit RNase inhibitor will inhibit 50% of 5ng of RNase A and requires 
1mM DTT so you appear to be okay so far...

> Does anyone has similar experience with RNase inhibitor? I wonder whether this 
> is a batch of bad RNase inhibitor from Ambion.  

Maybe the problem is your storage conditions.  RNase inhibitor is subject 
to denaturation and since it binds to RNases, denaturation can actually 
result in the release of RNases.

> With peptide inhibitors the dangerous part of your procedure is the
> moment when you add the phenol/chloroform- if there's a lot of RNase
> present, and if the inhibitor denatures slightly faster than the RNase
> (which is not unlikely), you've got trouble.  
>
> Steve LaBonne *********************** (labonnes at csc.albany.edu)

Also the RNase A may be renaturing after the phenol/chloroform.  We 
usually include a proteinase K digestion prior to extraction to eliminate 
RNAses.

> I suggest contacting Ambion about this problem (techserv at ambion.com). Their
> products have never given me any problems, and their techserv has always
> been excellent, in my experience.
>    Tracy 

I echo these sentiments.  I also have found Ambion techservices and their 
manuals to be extremely helpful.

Rich Rohan
University of Maryland
rrohan at umabnet.ab.umd.edu





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