hi,
I think I am getting primer dimer for my reference gene (small peak in the no template control) and for some reason this peak increases when I add in template. What is the reason for that? Also, it only seems to occur when my template is diluted. Contamination maybe?

Thanks,
F.

We have been discussing this in our lab for awhile now... in most cases our amplification dependent "primer bands" are not dependent on template, but we have seen this phenomenon as well...

We also see a depletion of this "primer band" in the presence of increased template in all cases...

In the case where it is not in the water control but only appears in samples with small amounts of template we are still debating what this means, for now I think of these as "template-dependent primer-dimers" although the idea that they are "primer dimers" is not confirmed...

What I am trying to say is that this seems to be a common phenomenon that isn't well explained and I don't think it represents contamination but it is a problem for some Real time PCR applications (particularly if syber green is used)

HTH

I have also seen this, with SYBR green qPCR

I have always attributed it to the change in efficiency of the PCR reaction as the template value changes. When I first set up and ran my standard curves and determined my efficiencies to use the delta delta Ct method for analysis, I noticed that template amount made a large difference for some amplicons. For example, if I tested 5X, 2X, 1X, 0.5X, etc...a large range of template dilution...I found that good efficiency only really happens across a certain part of the range of template. Outside that range, primer-dimers and non-specific products were evident. This is why I'm always harping at people on here to maximize their qPCR efficiencies

anyways, I don't know the exact reason, but I always attributed it to lack of reaction efficiency, dependent on template amount...becca, I like your name better though