User manual
Droplet Digital
™
PCR Applications Guide | 17
Designing Droplet Digital
™
PCR Experiments
The example is relevant to any gene that is present at the normal rate of
2 copies/diploid genome, such as RPP30, and provides a basis for a digital
screening experiment to determine the optimal digital range.
For sample DNA loading, follow these guidelines:
■
Assess input DNA/RNA concentration using A
260
spectroscopy to ensure the target
DNA/RNA concentration is being loaded within the dynamic range of detection
Note: An A
260
measurement depends on various factors and does not distinguish
between intact and fragmented targets. As such it provides only an estimate of your
DNA concentration.
■
Add no more than 1 µg of digested DNA to the 20 µl reaction (final concentration
of 50 ng/µl)
■
Intact DNA requires restriction digestion for optimal performance, especially at a
concentration above 3 ng/µl (60 ng/20 µl reaction). For copy number and absolute
quantification, always do restriction digestion at all DNA concentrations unless you want
to access proximal replicate sequences
■
Do not perform a restriction digestion of the DNA sample within the amplicon sequence
■
Fragmented DNA (for example, formalin-fixed, paraffin-embedded [FFPE] samples) may
not need restriction digestion. However, some assays and/or targets require digestion
for optimal target detection regardless of fragmentation
■
cDNA does not require restriction digestion
ddPCR Experimental Workflow
Droplet Generation
For droplet generation, transfer 20 µl of a PCR reaction containing sample nucleic acid,
primers (and probes for a TaqMan experiment), and the appropriate Bio-Rad ddPCR
supermix to the middle rows of a DG8
™
cartridge for a QX100 or QX200 droplet generator
(Figure 2.4). We recommend creating an initial reaction pool that is slightly more than 20 µl
(22–25 µl) to ensure that 20 µl of mixture is transferred to the DG8 cartridge. Reaction
mixes should be combined and well mixed in a separate tube and not in the droplet
generator cartridge. Reaction mixes should then be transferred to the DG8 cartridge
already preloaded in the DG8 cartridge holder.