User manual

Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting
86 | Droplet Digital
™
PCR Applications Guide
The temperature-sensitive assay used to generate Figure 9.7 is relatively long and very
GC-rich (244 bp, 74% GC). If it is suspected that the variation in concentration is due to
thermal cycler performance, consider:
■
Increasing the hot start from 94°C for 10 min to 96°C for 10 min
■
Raising the denaturation temperature from 94 to 96°C for the first 5 cycles
■
Purchasing Bio-Rad’s C1000 Touch thermal cycler with 96–deep well reaction module
■
If drops in concentration estimates are consistently confined only to a quadrant(s) of the
block, contact the manufacturer and request thermal-couple uniformity analysis and,
if necessary, repair
Concentrations Consistently Lower than Predicted
If concentrations measured in ddPCR are consistently lower than predicted, consider
poor target accessibility, poor or incorrect assay design, or the presence of PCR inhibitors
in samples.
It is possible that the reference concentration measurement that suggests ddPCR
concentration calls are low is, in fact, in error and is reporting a higher than actual
concentration. ddPCR gives a concentration measurement of intact DNA targets while
spectroscopic measurements typically do not distinguish between degraded and intact
nucleic acids.
Also consider the following options:
■
Make sure the ddPCR assay has been optimized by running a temperature
gradient experiment
■
Amplicons longer than 150 nucleotides may require longer annealing times during PCR
■
If duplexing 2 assays together for the first time, test them in a singleplex assay using
the same sample to confirm that the assays are not interfering with one another
■
Verify that the fluorophore is not conjugated to a G residue
■
Add the recommended primer (900 nM) and probe (250 nM) concentration