PhiX-174 virion DNA (New England Biolabs) is mixed with PCR reagents containing 1X Platinum Multiplex PCR Master Mix (Life Technologies), 200 nM probe (IDT), 1 μM forward primer (IDT), 1 μM reverse primer (IDT), 0.5% (v/v) Tween 20 (Sigma-Aldrich), and 2.5% (w/v) Poly(ethylene glycol) 6000 (Sigma-Aldrich). The reaction mix is printed with a PDC70 capillary into 100 μL HFE 7500 oil with 5% (w/v) PEG-PFPE amphiphilic block copolymer in a 0.2 mL PCR tube. After printing, the oil is replaced with 50 μL FC-40 oil (Sigma-Aldrich) with 5% (w/v) PEG-PFPE amphiphilic block copolymer. The emulsion is amplified using the following program on a Bio-Rad T100 thermocycler: 2 m 30 s at 95 °C; 35 cycles of 30 s at 95 °C, 1 m 30 s at 60 °C, and 30 s at 72 °C; and a final extension of 5 m at 72 °C. The emulsion after thermocycling is imaged on the EVOS Cell Imaging System in brightfield and GFP channels. Intensity data is extracted from each droplet; coalesced droplets with a diameter greater than 80 μm were excluded from analysis. The Poisson estimator was calculated from the observed fraction of positive droplets by the following equation: 23 (link).
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