3kb PCR amplification

Kimberley Snowden ksnowden at hort.cri.nz
Thu Mar 15 15:44:38 EST 2001


There a quite a few things you can try to get difficult RT-PCRs to work.
I had a long (15kb) message I had to work with, and what I found helped
was:
-Don't use a one step RT-PCR, instead do your first strand seperately,
and add some to a PCR.
-use a good RT enzyme - I like Superscript II (no affiliation)
-sometimes different incubation temperatures for the RT step can help.
For my transcript, if I did the RT step at 42 degrees I got little to no
amplification, but everything worked fine when I did the RT step at
elevated temperatures (up to 55 degrees).

Hope that helps.

Kim




Claudio Monetti wrote:
> 
> How can I amplify a 3.7 kb PCR fragment from total cDNA?
> 
> --
> 
> Claudio Monetti
> Laboratorio di Biologia Cellulare
> Dipartimento di Biologia Strutturale e Funzionale
> Via Dunant 3
> Varese
> Tel 0332-421328




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