RT-PCR PROBLEMS

[suihuang at usa1.com]

In article <4cgj43$eq at mserv1.dl.ac.uk>, ANDREI.POPOV at bbsrc.ac.uk 
says...
>
>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
>

That's right.
but if you don't want to purchase superscript or other thermosatble 
rev transcriptase and still like to use specific primers for the RT 
step, you might use you original 3' primer for RT and use a second 
NESTED 3'  primer (i.e. upstream of your RT primer) for your PCR. this 
also circumvents the problem of mispriming to non-specific sites.

Sui Huang
Children,s Hospital Boston