One of the very first things you need to do when getting set up for quantitative PCR (qPCR) is to determine the efficiency of the assay because knowing the assay efficiency is critical to accurate data interpretation. And you have to do this every time you design and purchase a new primer pair.
Ideally, the efficiency of the assay should be 100%. This means that during the logarithmic phase of the reaction, the PCR product of interest is doubling with each cycle. Perfect PCR efficiency will demonstrate a change of 3.3 cycles between 10 fold dilutions of template.
Sometimes the efficiency is below 100% and sometimes you can get readings of PCR efficiency above 100%. In a future article, we’ll discuss what causes high and low PCR efficiencies and how to fix it.
Today’s article is about the important considerations when setting up a PCR efficiency test for your new assay. The three key ingredients to focus on when getting started are the type of template, the primers, and the chemistry.
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