RT-PCR PROBLEMS

Andrei Popov ANDREI.POPOV at bbsrc.ac.uk
Thu Jan 4 08:01:55 EST 1996


Hello all,
>
>I have been trying to amplify messages using RT-PCR, but I don't get any
>bands after the PCR.  I failed to see bands even with primers from
>Clontech designed to amplify G3PDH.  The protocol that came with the
>primers recommended that I use oligo-dT or random primers during the RT
>step, but I just used the 3' PCR primer.  Has anyone out there have
>success with RT-PCR without using oligo-dT or random priming?  

Hello there,

it does not work. 
I (and others) tried it before.
The reason is very simple- your PCR primer is just too long
and anneals everywhere at the tempr of rev transcription.
There are some hints:

1. Try rev transcription at 50 C- SuperScript RT
   survives at 50C
2. Reduce the conc of the primer during rev transcription
3. If 1 and 2 do not work you have to use oligodT or
   (as we did) design a short specific antisense primer
   with Tm around 37-40 C.


best wishes

Andrei




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