RT-PCR Troubleshooting - HELP!

Adam Dempsey adam at tcgu.med.utoronto.ca
Mon Jun 1 11:34:28 EST 1998


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
adam at tcgu.med.utoronto.ca.nospam

please remove the nospam to send me email.



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