Removing any traces of DNA

David L. Haviland, Ph.D. haviland at KIDS.WUSTL.EDU
Thu Dec 14 14:03:33 EST 1995


On Thu, 14 Dec 1995, Jean Peluso wrote:

> I would like to know how I can remove completely DNA from a preparation
> of in vitro synthetised RNA. I have already tried the DNAse I recommended
> by Invitrogen (T3 Transcription Kit), but when I perform RT-PCR on the
> RNA  preparation I unmask traces of DNA, even by using more DNAse and
> prolonging the incubation time of the reaction. The "highest" 
. conditions I have tried in order to degrade one microgram of DNA were: 
. 30 min @ 37!C with three units of DNAse. 
> 
> Any help would be welcome.

Jean:  When I was doing RNAse protection assays, I routinely used 20U of 
RNAse free DNAse I in my protocol (Harvard manual) to completely digest 
my 1 ug of starting template.  I once set up the reaction with cold 
nucleotides and took aliquots and ran them on an agarose gel.  At 37'C in 
5 mins the template was gone (visually).  The protocol called for 30'mins 
at 37'C.

Hope this helps,
David

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