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.
Tracy
