RT-PCR technique

Jonathan Shillingford jmshillingford at hotmail.com
Tue Mar 3 18:40:53 EST 1998


Hey again,

Part two! I have used the Promega Access RT-PCR kit, with minor
modifications, for my RT-PCR experiments. I have done several reactions
mixing and matching buffers, RTs and differnt DNA polymerases (Tfl, Taq,
Tth) to come up with the best combination. 

Are there any inherent advantages to a two-step RT/PCR method (e.g.
SuperScript RT followed by Taq PCR) as opposed to a one-step RT-PCR
method (e.g. AMV-RT/Tfl combination)? Which would you recommend? I am
not particularly concerned with generating full-length transcripts,
hence I use AMV-RT and gene-specific primers as opposed to SuperScript
and an oligo(dT)n primer.

Any help/advice/support is much appreciated


Jonathan Shillingford



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