RNase inhibitor does not work ???

Tracy Aquilla aquilla at salus.med.uvm.edu
Wed Feb 8 14:47:22 EST 1995

In Article <zxiong.84.04AE9CF2 at ag.arizona.edu>, zxiong at ag.arizona.edu
(Zhongguo Xiong) wrote:
>Hi, netters:
>I have always taken as granted that RNase inhibitors, whatever sources they 
>come from, will work to inhibit RNase activity. An experiment I run yesterday 
>showed that is not true. Because some of our experiments were not working, I 
>set up to test the RNase inhibitor activity. 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. A 
>reaction without RNase but otherwise identical was done as a control. After 90 
>minutes of incubation, the solution was extracted with phenol:chloroform:IAA 
>and precipitated with ethanol. The agarose gel electrophoresis afterward 
>showed that RNase completely destroyed my RNA even in the presence of the 
>RNase inhibitor. The control lane showed no degradation of RNA at all.
>Does anyone has similar experience with RNase inhibitor? I wonder whether this 
>is a batch of bad RNase inhibitor from Ambion. Could RNase inhibitors from 
>other sources (Promega, BRL, stratagene) perform better in a similar test?
>Any comments and suggestions are welcome.
>Zxiong at ag.arizona.edu

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.

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