RT-PCR

[Jinling Xie]

In article <2polu4$ajr at network.ucsd.edu>,
Jonathan Hecht  <jhecht at ucsd.edu> wrote:
>In article <leach-260494212126 at ppp-8-28.bu.edu> Martin Leach,
>leach at mbcrr.harvard.edu writes:
>>BTW if doing RT-PCR - DNAse treat your RNA before RTing it. Otherwise
>>unless u r pcring over an intron (in the DNA) u won't know whether the
>>product is real or due to DNA contam.
>
>As well DNAse treating the RNA, its important to do a control PCR
>reaction using mock-reverse transcribed RNA as a template (do this by
>adding everything to the RNA except reverse transcriptase).  That way you
>will be absolutely sure you have no DNA contamination.  For a good
>reference about this, see the SRY1 cloning paper (Goodfellow, I think).
>
>Jonathan


I generally include a negative control in my RT rxn in which I add
RNAase A treated RNA instead.  You should not get anything from this
control when pcring.  If you do :-( ===>  DNA contamination!!!!!

--Jinling