RT-PCR question

[Richard R. Hardy]

> Adi Santoso (santoso at badlands.NoDak.edu) wrote:
> : i try several time RT-PCR with 3' primer using oligo dT (16 mers) just 
> : never work. I check all the reagents and template RNA, they are all 
> : oke...!!! any suggestion for me.  I just almost give up with it..
> 

Try doing your RT reaction with random hexamers and then the PCR with a
specific 3' primer.  For big transcripts, we sometimes get low signals
with oligo dT, even doing the PCR with specific 5' and 3' primers (when
they're located 5' in the gene).  Even if Oligo-dT works well for the RT,
it still may not be a very good primer for the PCR (GC content of 0!)...

-- 
R. Hardy
Member, Institute for Cancer Research,
Fox Chase Cancer Center, Philadelphia, PA
(215) 728-2463