User manual

88 | Droplet Digital
™
PCR Applications Guide
Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting
High-Fluorescence Amplitude Droplets
Droplet coalescence can create droplets that are much higher in fluorescence amplitude
than the other positive droplets. This can be caused by poor droplet transfer technique
or extended storage of the droplets pre– or post–thermal cycling. Adjust the scale on the
1-D or 2-D amplitude charts in order to set the thresholds in these cases.
Troubleshooting EvaGreen ddPCR Reactions
EvaGreen binds to both double-stranded DNA and, to a lesser extent, nonspecifically to
single-stranded DNA. Therefore, adding excessive amounts of primer or DNA starting
materials to EvaGreen ddPCR reactions can result in an inability to distinguish positive
droplets from negative droplets. The separation of positive and negative fluorescence
can be affected by total added DNA, including primer concentrations, in addition to the
previously discussed fluorescence amplitude differences in amplicon length or amplicons
with varying PCR efficiencies.
For good separation, a combined primer concentration between 75–250 nM is
recommended and 100–200 nM is optimal (Figures 9.9A–B). Figure 9.9C is an example
of the effect of EvaGreen ddPCR with 900 nM primers on increasing concentrations of
DNA that should be negative for the primer set added. Figure 9.9D shows the primer
concentration is decreased to 100 nM and the negatives are returned to a uniform cluster
of droplets. Similarly, adding more than 130 ng DNA/20 µl reaction can cause the negative
droplets and positive droplets to have poor separation. Figure 9.10 shows an experiment
with 100 copies/µl mutant DNA spiked into increasing amounts of wild-type DNA. As the
total added DNA concentration increases, the positive fluorescence amplitudes decrease
and the negative fluorescence amplitudes increase.