AAT Bioquest

What should I consider when I set up a PCR efficiency test for my assay?

Posted May 12, 2023


There are three key factors you need to consider when setting up a PCR efficiency test for your new assay - the type of template, the primers, and the chemistry.

Type of Template

Your PCR template can be one of a variety of nucleic acids, including DNA oligos, genomic DNA, plasmids containing the gene of interest, or the PCR product of interest. 

For the standard curve, the template should be quantified and diluted in at least 5 dilutions with a 1:10 dilution ratio for the widest linear range. Using 1:4 dilutions may work but you should know that it limits the range within which you can be sure that the primer pair is accurate. 

Duplicates of each template dilution are the minimum requirements. Triplicates can increase confidence in the data, especially if, for some unknown reason, one data point drops out.  

Quantifying and diluting the template accurately is crucial for obtaining accurate and reliable results in your PCR efficiency test.


It’s important to check the efficiency when using a new pair of primers for qPCR. Do this even if you are using primers that you know work well but they are from a new batch. Never work under the assumption that primers from different batches will work at the same efficiency. Sometimes they don’t. 

The concentration of primers to use depends on the enzyme chemistry and starting with the recommended concentration from the kit supplier is a good idea. Too much primer can lead to dimer formation, while too little primer can comprise the efficiency. 

If optimizing and modifying the primer concentration doesn't improve efficiency, it may be necessary to redesign the primers. The desired level of assay efficiency depends on the sensitivity you need for accurate quantification. If most of the data falls between cycles 18-30, an assay with 80% efficiency may be acceptable, but if you require accurate quantification beyond cycle 35, you need the most efficient assay possible.


Always re-check the assay efficiency when buying a new lot of kit to ensure that the kits do not vary. 

If you're using SYBR Green assays, it's important to include melt curve analysis to detect the presence of primer dimers, which can affect a hydrolysis probe and reduce efficiency. If efficiency is low, you may need to run the reaction on a gel to analyze what else may be amplifying in the reaction. 

For probe assays, probe design and compatibility of three primers in the mix are also important considerations. If you suspect efficiency problems are due to the probe, you can run your primers with a SYBR Green kit to determine if they work well. If they don't, you need to optimize your primers.

Additional resources

Validating Real-Time Polymerase Chain Reaction (PCR) Assays

Polymerase Chain Reaction (PCR)

ROX Reference Dye *50X fluorescence reference solution for PCR reactions*