HELP. Multiplex RT-PCR with GAP as a housekeeping gene.

Andy fedoriw at lalaland.mail.med.upenn.edu
Wed Feb 14 10:27:03 EST 2001


I think the trick would be to establish conditions (e.g., a lower primer
concentration, higher annealing temp, etc.) in which your current primers 
hit their linear range at 30/35 cycles. 

I had the same problem, and had to lower the concentration of the primers
for my housekeeping gene 10-fold. I got co-amplification, but there was
still a bias towards my control product vs. the amplicon of interest.

hope that helps... andy

In article <3A8A850C.2D6818A3 at utanet.at>, Thomas Mohr
<thomas.mohr at utanet.at> wrote:

> Dear Colleagues,
> 
> currently we're trying to establish a multiplex RT-PCR with GAP as a
> houskeeping gene. Intended is the useage GAP primers plus a second
> primer pair (and maybe third). However, our GAP products require only
> 22-25 cycles, compared to 30 to 35 cycles of the others. Coamplification
> with GAP primers added after 10-15 cycles lead so far to an almost
> complete quenching of the other product. 
> 
> Does anybody know of GAP primers fulfilling following condditions:
> 
> Annealing temperature 55-60C, required cycles: 30-35 ?
> 
> Thanks for your help !
> 
> Thomas






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