User manual

Droplet Digital
™
PCR Applications Guide | 15
Designing Droplet Digital
™
PCR Experiments
Fig. 2.3. Assay design output.
Several important design features are not addressed by Primer3Plus (or the Primer3 MIT site).
■
To ensure primer specificity, use tools such as BLAST (Basic Local Alignment Search
Tool), hosted at the National Center for Biotechnology Information (NCBI), either in
the “traditional” general search form (www.ncbi.nlm.nih.gov/BLAST) or a form
tailored specifically to check that PCR primers (http://www.ncbi.nlm.nih.gov/tools/
primer-blast/) match only your intended target
■
Check that common SNPs do not land in your primer sequences
■
Check for the secondary structure of the amplicon using the Mfold program
(http://mfold.rna.albany.edu/?q=mfold). Primer binding sites should be predicted
to be “open” (that is, not base-paired in a secondary structure) at the PCR
annealing temperature
Sample Preparation
The quality of the nucleic acid preparation from the sample of interest can impact ddPCR
results. An optimized protocol should be used to extract the DNA or RNA from the raw
material you are testing. Ensure that the sample has not been degraded, for example,
by heating above 60°C. Although some PCR inhibitors are less detrimental to quantification
accuracy in ddPCR than in other technologies, we recommend removing as many of these
as possible during the nucleic acid purification phase. If known inhibitors cannot be readily
removed, consider reducing their impact on the PCR reaction by diluting the sample 1:10.