Fig 4. Influence of cycle number, elongation and denaturation times and ramp rate.

(a) With a higher number of cycles in the PCR, separation of the positive and negative droplets is more distinct and the fluorescence level of the positive droplets higher. (b) Lower ramp rate (2°C/s or 1°C/s) promotes better droplet separation as well as longer elongation or denaturation steps do (unless indicated otherwise, one minute elongation, 30 seconds denaturation and a ramp rate of 2°C/s was used). ddPCR was performed with EGDe DNA (≈ 10³ copies/sample).