How to remove DNA from RNA for RT-PCR

brett brett at BORCIM.WUSTL.EDU
Wed Sep 3 19:27:04 EST 1997


>I've treated my RNA with RNase-free DNase I (following Sambrok et al.,
>and Ausubel et al. procedures) at least 3 consecutive times and I still
>get DNA-generated bands in my RT-PCR. Does anybody know a method to
>absolutely remove DNA contamination from RNA preparations?

Yes, this is indeed a problem. Most people don't use sensitive DNA detection
methods when checking their digestions as we have.  In fact someone in this
lab is testing various nuclease combinations to obtain the best conditions
for
cleaning up RNA samples. She isn't finished, but she tells me 3 sequential
digestions using cocktails of DNase I *and* ds- and ss- Exonucleases from 
Epicentre drop the DNA titer 7 orders of magnitude by qcPCR, with good recovery
of RNA biologic activity. 


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




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