Primer optimization (with probes) - Urges... (Jul/16/2008 )
I am new to qPCR and have a question regarding the primer optimization step. I have primers and TaqMan probes, but in literature I alway read that the primer optimization goes WITHOUT the probes (so with SybrGreen?). Do I really have to skip the probes when doing my optimization matrix? Or shoud I include the probes in this step?
You might wish to check the AB guidelines on the topic.
As you see, they recommend to first optimise your primer concentrations with 250 nM of your probe (which gives satisfactory robustness) and then optimise the probe concentration.
However, if money is an issue for you, I dont think that you could go wrong if you optimise primers with SYBR Green.
We optimized with the probe. Each primer is optimized 100, 300,500,700 & 900nM with 400nM of the probe, then the probe is optimized 100, 200, 300 & 400nM with 900nM of the primer . 10 tubes total counting the non template control and double so the assay is made duplicate.