Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 22)

16 | SsoAdvanced ™ Universal SYBR ® Green Supermix Instruction Manual 16 |
PCR Performance Not 100% Efficient
If you have already ruled out your samples as a source for poor efficiency, then the assay may
be the cause of the problem. Please review the section on assay design in this manual for
further information (page 3).
Also, consider the following corrective action:
Perform a temperature gradient experiment to determine the optimal annealing temperature.
Set up the gradient as follows:
a. Use several representative samples in your project.
b. Set the temperature range 10ºC above and 6ºC below the calculated annealing temperature.
c. Choose the final annealing temperature based on overall performance related to specificity.
If the PCR efficiency is >100%, and you have additional peaks in your melt profile around
the mid-70s (see Figure 13), this is most likely primer dimer. Consider the following
corrective action:
Compare the NTC well melt curve profile to all other wells that contain samples. If the sample
wells and NTC wells exhibit primer dimer, then the melting temperatures will align. Simply
stated, a balance of primer to template is required when using SYBR
® Green assays. When
too high a primer concentration is used and the primers are prone to dimers, primer dimers will
form at low and no sample concentration inputs.
To correct this, perform a primer matrix analysis for every primer pair prior to the first time you
use them in your project to ensure the correct final primer concentration is selected. Table 5
illustrates a sample plate layout for a primer matrix, where the forward and reverse primers
are plated at various final concentrations. (Note that you can build a matrix as large as you
prefer.) Load the plate with a small amount of sample (expression-dependent) into every well
and perform a real-time PCR run. Choose the primer concentration that exhibits the following:
lowest Cq value, highest fluorescent signal, and no primer dimers. Note that the final primer
concentrations do not have to be equamolar.
Fig. 13. Primer dimers at –79ºC.
Melt Peak
-d
(RF
U
)/d
T
Temperature, Celsius
300
200
100
0
80 85 90 95 75 70 65