User manual

Droplet Digital
™
PCR Applications Guide | 11
2 Designing Droplet
Digital
™
PCR Experiments
Assay Design for Droplet Digital PCR
As with any PCR-based technology, assay design and sample preparation are important
for obtaining high-quality data. Before running a Droplet Digital (ddPCR
™
) experiment,
know the goal or possible expected outcomes of the experiment because different types of
experiments require different controls, sample preparation, amounts of DNA or RNA, and
data analysis.
The amplification reaction of target molecules in ddPCR workflows follows similar principles
of real-time PCR.
■
Plan to amplify a 60–200 bp product
■
Avoid regions that have secondary structure when possible
■
Choose a region that, ideally, has a GC content of 40–60%
Designing Primers
Widely accepted quantitative PCR (qPCR) design guidelines apply to ddPCR primer
design. Important criteria for single primers include melting temperature (T
m
), length, base
composition, and GC content. In addition, because primers are used in pairs, ensure that
paired primers do not exhibit significant complementarity between 3' ends because this
can result in primer-dimers. Extensive primer-dimer formations can significantly decrease or
prevent amplification. The QX200
™
Droplet Digital PCR system will support both hydrolysis
probe (TaqMan) and DNA binding dye (EvaGreen) assays. All information in this chapter
applies to both types of assay, except for the Designing Probes section.