User manual

Droplet Digital
™
PCR Applications Guide | 23
Designing Droplet Digital
™
PCR Experiments
Fig. 2.10. Thermal gradient optimization.
PCR Optimization Using Thermal Gradients
Optimizing the annealing temperature of your PCR assay is one of the most critical
parameters for reaction specificity. Setting the annealing temperature too low may lead
to amplification of nonspecific PCR products. On the other hand, setting the annealing
temperature too high may reduce the yield of a desired PCR product. Even after calculating
the T
m
of a primer, you may need to determine the optimal annealing temperature empirically
because this temperature depends on buffer conditions as well as primer and probe
concentrations. This involves repeating a reaction at many different temperatures.
Similar time-consuming tests may also be required to optimize the sample
denaturation temperature.
As with classical PCR reactions, the optimal annealing temperature for an assay can
be easily determined using thermal cyclers that have a thermal gradient feature.
The C1000 Touch
™
thermal cycler offers a gradient feature. The gradient feature enables
you to test a range of temperatures simultaneously, optimizing the annealing temperature
in a single experiment.
To find the optimal annealing temperature for your reaction, test a range of temperatures
above and below the calculated T
m
of the primers. The optimal annealing temperature is
the one that results in the largest fluorescence amplitude difference between the positives
and negatives and that avoids nonspecific amplification. It is also important to view the
concentration values obtained across the thermal gradient simultaneously when considering
the fluorescence amplitude separation of positives and negatives. A sample annealing
temperature optimization experiment is shown in Figure 2.10 demonstrating separation
of positives and negatives of replicate samples across the thermal gradient.
For two-color assays, a thermal gradient can be used to identify a temperature where both
assays perform well.
0 20,000 40,000 60,000 80,000 100,000
Event number
Channel 2 amplitude
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
A01 B01 C01 E01D01 F01 G01 H01
65°C 55°C