Good Rnase free DNase sources?

brett brett at BORCIM.WUSTL.EDU
Wed Oct 1 09:01:30 EST 1997


>S. MORI (smori at NMSU.Edu) wrote:
>: Dear molbio's
>
>: I am trying to get a hold of some good RNase free DNase... I had some 1
>: year old Stratagene stuff, but the freag'n thing ate all my RNA... It had
>: been stored at -20C... It may have been the age (I doubt it) but I don't
>: wanna risk it again.... anybody has know of a better source?
>
>I've always had good results with the DNase I from GibcoBRL - the RNA prep
>is immediately usable in RT-PCR, and I assume, is good enough for
>northerns (although I don't usually bother for northerns).
>
>Cheers,
>--
>Dave Pearton
>pearton at u.washington.edu

Dave, have you ever shown that your RT-PCR is free from DNA by doing the RT-
control?  While we find that DNAse I and RQ1 DNase do indeed destroy DNA, it
is not nearly quantitative.  That is, =>0.1% of DNA remains by qPCR (RT-).
Someone in our lab has been trying various combinations of nucleases and is
now able to decrease the DNA titer to undetectable levels, but not with only a
single step.  I suppose then it depends also on the sensitivity of your specific
PCR (ours can detect 1 molecule DNA ~30% of the time).  I'd be glad to hear your
suggestions.



Brett Lindenbach
                             
Washington University - St Louis                  
brett at borcim.wustl.edu                             




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