rt-pcr problems

Simon Dawson Simon.Dawson at nott.ac.uk
Wed Jan 3 03:29:35 EST 1996


William Sun wrote:
> 
> 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?  I don't
> understand why random or oligo-dT priming would give better results.
>

Hi,
   I'm not sure of any reason why it wouldnt work other than the fact people 
always say to do the RT step w/ dT or p(dN)6. I assume you checked that the 
3' PCR primer is _antisense_ with respect to the mRNA template? I don't know 
how Clontech have their primers set up, but off the top of my head it seems 
more logical for the 5' PCR primer to be the antisense one wrt the mRNA. 
Just an idea.

        Simon.

-- 

Dr. Simon Dawson       TEL:+44 (0)115 9249924 ex. 44789
Dept. of Biochemistry  FAX:+44 (0)115 9422225
Queens Medical Centre  Email:Simon.Dawson at .nott.ac.uk
Clifton Boulevard      http://www.ccc.nottingham.ac.uk/~mbzspd/Simon.html
Nottingham
U.K.                   "Back off man, I'm a scientist!" - Bill Murray.



More information about the Methods mailing list