How do you determine the efficiency of a real-Time PCR assay?

Ideally, the number of molecules of the target sequence should double during each replication cycle, corresponding to a 100% amplification efficiency. Similarly, if the number of replicated molecules is less than double this is due to poor efficiency – below 100%. The most common reasons for lower efficiencies are bad primer design and non-optimal reagent concentrations or reaction conditions. Secondary structures like dimers and hairpins or inappropriate melting temperatures can affect primer template annealing which results in poor amplification. Since each additional dilution contains appropriately lower starting amounts of DNA, differences occur between cycle threshold (ct) values in serially diluted samples (see below).



One way of calculating the amplification efficiency is by making serial dilutions of your target. Once you obtain their Ct values, plot them on a logarithmic scale along with corresponding concentrations. Next, generate a linear regression curve through the data points and calculate the slope of the trend line. Finally, efficiency is calculated using the equation: \(E=-1+10^{left(frac{-1}{slope}ight)}\)

Or use this calculator which does the work for you. Be sure to understand what influences the slope of the amplification curve, as it can otherwise be misleading.

Typically, desired amplification efficiencies range from 90% to 110%. The theoretical maximum of 100% indicates that polymerase enzyme is working at maximum capacity.


It's a destabilizing agents, which is required to loosen the bonds holding the DNA molecule together. It does this by competing effectively with the H-bonding between the base pairs.


If this question is based on a lab you did, I can't answer your question. What's the name of your lab? Maybe I can look it up and figure this one out more effectively.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.

Did you do such a thing? Maybe just write what I wrote above and it'll be fine.

Good luck

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