Inactivation of DNase I

AK via methods%40net.bio.net (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 
etc.
hth
AK
"Gouttesoulard Marina - Saint-Beauzire " <marina.gouttesoulard from lonza.com> 
wrote in message news:mailman.385.1213106791.3533.methods from net.bio.net...
Hello,
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.


Marina
> mailto:marina.gouttesoulard from lonza.com
> http://www.lonza.com
>
>

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