RT-PCR Troubleshooting - HELP!

Gail Otulakowski gotulak at sickkids.on.ca
Wed Jun 3 18:00:04 EST 1998


Adam Dempsey wrote:
> 
> Hello.
> 
> I have a few questions regarding RT-PCR.  Another member in my lab has
> performed RT-PCR using the rTth kit from Perkin-Elmer and the TITAN kit
> from Boehringer Manheim.  Using the rTth kit the appropriate band was
> amplified.  However, after attempting the same RT-PCR several months
> later all that was achieved was a big smear.  Using both kits resulted
> in a smear.  The same primers were used. There was no RNA degradation.
> Also, the funny thing is that sometimes the proper band does appear.
> 
> Another problem is that the primers were designed within the coding
> region.  When genomic DNA was amplified the same 800nt fragment was
> obtained.  It is possible that there is no intron here, correct?
> 
> Thank you.
> 
> Adam Dempsey

You are correct, if there is no intron between the primers you will get
the same size amplified band from genomic DNA PCR and RT-PCR.  Do you
happen to know the gene structure?
As far as your problem that a previously successful RT-PCR fails to work
several months later, sometimes primers go "bad" - try having new ones
synthesized.

Gail Otulakowski



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