PCR efficiencies

There are several ways to determine PCR efficiencies. The easiest method is to make a dilution series of the PCR product of interest. (If you are performing RT PCR, the PCR product should be from cDNA and not genomic DNA, so that the probe matches the sequence). The dilution series should cover at least three orders of magnitude (1 to 1000x). For example

DilutionRelative concentration= Template, T
1/10001 x
1/500 2
1/200 5
1/100 10
1/50 20
1/20 50
1/10100
1/5 200
1/2500
straight1000 x
These are each used in a real time reactions. Once you run the real time reactions, you can set the threshold and have the machine determine the critical cycle # for each template concentration, T.

In the exponential phase of PCR, the fluorescence, R is related to PCR efficiency, E and cycle number, n by

Rn = k T (1+E)n
where k is just a constant which depend on the PCR machine, illumination intensity, light collection, etc. At threshold, the equation becomes:
Rth = k T1 (1+E1)Ct1
If we take the natural log of both sides we get
ln(Rth) = ln(kT) + Ct ln(1+E) = constant
Rearranging then we get:
lnT = constant - Ct ln (1+E)
So, for the dilution series data, if you plot ln(T) where T is relative template concentration, versus Ct you should get a line. The slope of the line is -ln(1+E). So you can solve for E from this slope. Typical values for E are 0.8-0.95.