Inactivation of DNase I
(by akhan357 from sbcglobal.net)
Tue Jun 10 15:04:52 EST 2008
I assume you are using DNase to remove DNA from your RNA prep to be used in
RT or real-time PCR. You can not heat RNA sample since 60C with divalent
cations will degrade RNA. so why bother to test this. in any case, since
Turbo DNase is so much more potent, perhaps a few molecules with residual
activity are doing this. why not revert back to regular DNase, if that will
produce sufficient chopping and subsequently can be removed/degraded
completely. In the past I have used Ambion's regular DNase free and its
removal with their kit (no heating involved) without any problems for RT-PCR
"Gouttesoulard Marina - Saint-Beauzire " <marina.gouttesoulard from lonza.com>
wrote in message news:mailman.385.1213106791.3533.methods from net.bio.net...
Have you solved your inactivation DNase problem?
I have also a problem in inactivation of DNase. I inactive DNase by heating
step 10min at 75°C. To check if the inactivation is efficient, I add 1ng DNA
in my inactivated sample and I compare the results obtained in PCR with a
control containing 1 ng DNA. There is a shift of 2 cycles with the
inactivated sample. So I think that the Dnase is not totally inactivated and
act on DNA added to destroy it. Have you got some solution to inactivate
Dnase without damage RNA.
Thanks a lot.
rq : the Dnase that I used was the Turbo Dnase from ambion and I already
test the inactivator of ambion's kit.
> mailto:marina.gouttesoulard from lonza.com
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