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Problem with QPCR - control gene does not amplify - (Jan/23/2006 )


I am having a problem with real-time PCR. I am using Sigma Jumpstart
Taq Readymix (cat no. D9191) and custom designed primers and probes
from Sigma Genosys. My reaction contains primers and a probe (FAM/TAMRA
linked) for my target gene, and primers and a probe (HEX/TAMRA linked)
for an endogenous control gene. In the past few real-time experiments,
we have had amplification of the target gene as normal (sometimes shaky curves are seen also though), however in some
(not all) reactions the endogenous control gene does not amplify.
Instead there is a very shaky amplification curve, that dips below
I realise that this may be a problem with the quality of our cDNA.
However, I was wondering if you know of any other possible explaination
for this?



what is the quality of your RNA template? and, I know that you are running primer/probe assays and may not do them, but if you have dissociation curves how do they look? they can be a good indicator of degradation


Wow, that qPCR data looks terrible. You don't even see a plateau...and the ct values look like they are all messed up.

What is wrong with your primers? Can you list them?