>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
>>Hope this helps,
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.
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