Removing any traces of DNA

brett brett at BORCIM.WUSTL.EDU
Thu Dec 14 14:21:09 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

The problem is that enzymes will find residual DNA sooner than your eye will.
Try gel purifying the RNA, redigesting with DNAse, and repurifying it. It sounds
like a lot of work, but you can get very clean RNA in this way. Our lab has done
this to make competitive RNA templates for quantitative RT-PCR.
Good luck, and write back if you need protocols.


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

"I own my own pet virus. I get to pet and name her." - Cobain




More information about the Methods mailing list