User manual

Droplet Digital
™
PCR Applications Guide | 83
Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting
No or Few Positive Droplets
If a new, never-before-tested assay fails to give positive droplets, consider the following:
1. The selected restriction enzyme may have cut within the target locus.
– Recommendation: test the assay against DNA digested with a different restriction
enzyme as well as undigested DNA
2. The target locus resides in a region that contains secondary structure.
– Recommendation: use restriction enzymes to cut the sequences surrounding
the region to be amplified in order to limit the number of possible interactions
with nearby nucleotides
3. The assay does not work at the predicted temperature.
– Recommendation: first perform an annealing/extension temperature gradient
to determine the temperature at which the assay works
4. The ddPCR reaction mix was not assembled correctly or the probe/primers
were not ordered correctly.
5. One of the assay components was designed incorrectly or a mistake was made
during synthesis.
No or Low Total Droplet Count
To determine your droplet count, select the well in setup, click Analyze, then click the
Events tab and make sure Total is selected. If the total accepted events or droplet counts
are less than 10,000 consider the following recommendations:
■
Use the recommended concentration of primer (900 nM), probe (250 nM), and
1x master mix. The QX100
™
and QX200
™
Droplet Digital PCR systems are compatible
only with Bio-Rad’s ddPCR supermixes. Using less than the recommended
concentration of any of these components may lower your droplet count
■
Load the DG8 cartridge with the appropriate volumes of sample and droplet generation
oil (20 µl and 70 µl, respectively). If less than 20 µl of sample is loaded, fewer droplets
will be generated. Be sure to load the sample before the oil
■
Use only purified nucleic acids. Any particulate matter (for example, residual fibers
from sample preparation columns or beads) in the sample should be removed before
assembling the ddPCR reaction mixture because these particulates can clog the DG8
cartridge’s microfluidic channels and disrupt droplet generation. To remove particulates
from purified nucleic acids, spin the sample at 10,000 x g for 1 min and transfer the
supernatant to a clean tube