OIL-PCR in tube-based format for master mix optimization

All steps were performed on ice or in a 4°C centrifuge until after emulsification. Fifty microliter PCR were prepared in a 1.5 ml microcentrifuge tube with varying experimental conditions. Two microliter of bacterial cells standardized to 10⁴ cells/μl were added to 48 μl of master mix and vortexed to evenly disperse cells before adding 300 μl of cold Droplet Generation Oil for Probes (BioRad 1863005). Emulsions were formed immediately after by shaking tubes at 25 Hz for 30 s on a Retch Mixer Mill MM 400 with adapters 11990 and 11993 (Mobio/Qiagen). Next, the emulsion mix was divided into four 70 μl aliquots in a PCR strip-tube and thermocycled as follows: 37°C for 5 min, 95°C for 10 min, then 38 cycles of 95°C for 5 s, 54°C for 30 s, and 72°C for 30 s, followed by final extension 72°C for 2 min. After PCR amplification, the aliquots were briefly vortexed and pooled into a clean 1.5 ml microcentrifuge tube. To break the emulsion, 50 μl of TE and 70 μl of Perfluorooctanol (Krackeler Scientific 45-370533-25G) were added and the mixture was vortexed vigorously for 30 s. Tubes were centrifuged at 5000 G for 1 min, and the upper aqueous phase was transferred to a new PCR strip tube and purified using AMPure XP beads as described below.

AMPure XP beads (Beckman A63880) were added at a ratio of 0.8 μl beads per 1 μl of recovered DNA, vortexed, and incubated for 5 min for DNA binding. PCR strip tubes were transferred to a 96-well magnet (Eppendorf Magnum FLX) to pull down beads for 5 min. Supernatant was removed with a multichannel pipette and the pellet was washed twice with 100 μl of 70% EtOH before drying for 10 min at room temperature. The bead pellet was suspended in 20–50 μl of TE and incubated for 5 min to elute DNA, before returning to the magnet and transferring supernatant to fresh PCR strip tubes. Eluted DNA was either run directly on a gel for qualitative analysis of amplification or used as template in qPCR assays.