Figure 4.

ddPCR assay with droplets prepared by PTE. (a) Fluorescence images of droplets after PCR amplification with TaqMan probes and primers for yeast genomic DNA templates at varying dilution factors. The fractions of observed fluorescence-positive droplets correspond with the template concentrations. (b) Scatter plot showing the size and fluorescence distribution from a sample in the dilution series. The population with low fluorescence (<20 AU) and small diameter (<30 μm) is composed of droplets containing no hydrogel particles (bottom-left). The population with expected diameter (30–40 μm) consists of single-hydrogel-core droplets. They form two tight clusters: high fluorescence (PCR-positive) and low fluorescence (PCR-negative, no-template droplets). (c) The average template copy number per droplet estimated by assuming a Poisson distribution scales with the controlled template concentrations over the tested three-decade range (R² = 0.9994 and error bar depicts standard error).