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Efficiency curve of primers

qPCR Pimers Curve

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#1 DionisioDD



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Posted 10 June 2019 - 05:28 PM

What happens is that I just acquired a couple of new primers, and the first thing I want to do is to check the efficiency of these primers with a curve using dilutions of .5ul, 1ul, 1.5ul and 2ul. The problem is that in the design of programs I do not know how to do it, my software is a StepOne Real-Time PCR Systems.
I plan to do it by quadruplicate, the first four as negative control, the next as positive and the next 16 with the different concentrations of primers.
I do not know how to place the various dilutions in the wells, I do not know how to configure it.
There should be something like the image, but in the concentrations that I mentioned and not the ones that appear


#2 OldCloner



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Posted 13 June 2019 - 04:21 PM

If I am understanding your question correctly, I think you want to use one standard concentration of your primers for all reactions, but vary the concentration of your target DNA, as you would do in any standard curve.  Carefully make a set of diluted template DNAs so you have  an accurate 1/10 dilution series. Plus a no-template control.  Then look at the r value for the curve you get.  I’m not sure why you want to change the conc. of the primers.

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