RNase inhibitor does not work ???
zxiong at ag.arizona.edu
Tue Feb 7 12:55:19 EST 1995
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
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