In article <01bf63ab$1636a2a0$41ba63ca at DNS.sj.he.cn>, Bethune
International Peace Hospital <genet at public.sj.he.cn> writes
>I use single tube RT-PCR protocol to detect GAPDH mRNA .
>the PCR products is a weak 309bp aim line and a strong line
>of 100bp(unexpected) or so . why? How to improve my system?.
>Any suggestions would be gratefully received!
Either it is primer dimer or the PCR enzyme is presumably extending any
falsely annealed primer during the low temperature 1st strand cDNA
synthesis. This extended primer is now a good match, but at the wrong
place, during the PCR stage.
If the PCR enzyme is Taq use anti-Taq antibody to inactivate it during
the cDNA step. If not, reduce the length of the cDNA step.
Optimise the PCR step better i.e. can you increase the annealing temp
to reduce any false priming. Are your primers really specific or are
they possibly quite short? Check for dimerization at the RT temp. Try
just doing reaction with no RNA. Do you see the approx. 100bp band? If
so should be primer dimer and it will be much less than 100bp.
If all else fails you may have to go to two step reaction or only add
one primer for the RT stage, pause the reaction and add the second
primer at the start of the PCR stage.
We do GAPDH with single tube and do not see a second product.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....