RT-PCR

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Thu Oct 8 23:33:44 EST 1992


G'Day Netters

I have some questions regarding PCR from RNA.  The standard method is to
use Reverse Transcriptase and oligi-DT to synthesise cDNA from total RNA
followed by standard PCR.  Another method is to use the dowmnstream primer
for the cDNA synthesis followed by PCR.  More recently Amersham has brought
out a taq polymerase called Tet-Z which has both RT and polymerase activity
(indeed Amplitaq from cetus does this also).  The reverse transcription and
the PCR is therefore done in the same tube in the same set of cycling
reactions, the first one being a 15-30 minute RT step followed by standard
PCR.  We have also tried these methods (when I say we, read 1 honours
student and a postdoc with little molecular experience, and I have limited
experience with RNA) and get extremely variable results.  Sometime we get
good results and othertimes no more than a smear.  Before I personally
start running some experiments to work this out I thought that I would ask
all of you out there in Netland for your opinion.

Any help will be appreciated.

Cheers, Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"If all else fails : Read the instructions"
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