Hi,
I am doing a qPCR and using a reference primer for my lab research project. However, I am not sure what is the purpose of having a singular primer for my reference gene and having both (reverse and forward) primers for my gene of interest. I would kindly appreciate your help.
Thank you
If it's a commercial reference gene primer set, it may simply be forward and reverse primers already mixed in a single pot.
You are correct: PCR with a single primer does not produce anything.
John Hildyard , you are not quite correct when you say that Victoria is correct saying that a single primer does not produce anything :)
Via specific priming, a single primer produces a ssDNA product. If there is no reverse priming, this product does not serve as template, so the amplification is linear, not exponential (what usually does not give analytically tractable amounts afetr 40-50 cycles). But when there is a mispriming event in the reverse direction, a dsDNA product is produced that has perfect binding sites at either end, and this is then eamplified exponentially:
http://rothlab.ucdavis.edu/protocols/semirandom.shtml
It should also be possible to use an RT primer with a binding site for the PCR primer, so that the cDNA contains two valid binding sites.
Well if we're going to be _that_ specific, fine. :P
Perhaps I should have said "would not produce anything of use for qPCR normalisation purposes"?
Victoria: what Jochen Wilhelm Jochen said.
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