Droplet-based Mutant Library Screening

DNA mutant libraries were diluted in 200 μg/ml yeast total RNA solution (Ambion) down to ≈8 template DNA molecules per picoliter. 1 μl of this dilution was then introduced in 100 μl of PCR mixture containing 20 pmol of each primer (Fwd and Rev), 0.2 mM of each dNTPs, 0.67 mg/ml Dextran-Texas Red 70 kDa (Molecular Probes), 0.1% Pluronic F68, 1x EvaGreen (Biotium), 5 U of DreamTaq^TM and the corresponding buffer (Fermentas). The mixture was loaded in a length of PTFE tubing and infused into droplet generator microfluidic chip where it was dispersed in 2.5 pl droplets (production rate of ≈12 000 droplets/s) carried by HFE 7500 fluorinated oil (3M) supplemented with 3% of a fluorosurfactant (29 (link)). Droplet production frequency was monitored and used to determined droplet volume, and pumps flow rates (MFCS, Fluigent) adjusted to generate 2.5 pl droplets. Emulsions were collected in 0.2 μl tubes as described before (29 (link)) and subjected to an initial denaturation step of 1 min at 95°C followed by 30 cycles of: 1 min at 95°C, 1 min at 55°C, 2 min at 72°C. Droplets were then reinjected into a droplet fusion microfluidic device.