Quantifying Phage Titers via qPCR

Phage titers in PANCE were estimated using qPCR as previously described.⁶⁶ (link) For each qPCR titer experiment, in addition to phage pools from evolution, a standard phage sample of a known high titer (1X10¹⁰ pfu/mL as determined by plaquing) was treated identically to create a standard curve. To titer a phage sample, eight serial 10-fold dilutions of phage were made into DRM (no antibiotics). 25 μL of each serial dilution was heated to 80°C for 30 min. Then 5 μL of heat-treated phage we combined with 44.5 μL of 1x DNase buffer and 0.5 μL of DNase (NEB). The DNase mixture was heated to 37°C for 20 min and then 95°C for 20 min to remove genomes from replication-incompetent polyphage. 1.5 μL of the heat-inactivated DNase mixture was pipetted into a 28 μL Q5 High-fidelity PCR reaction (NEB) containing SYBR Green (Invitrogen) and primers M13-fwd and M13-rev (see Table S6B). qPCR was run on a Biorad CFX96 Real Time system with the following cycling conditions: 98°C for 2 min, [98°C for 10 s, 60°C for 20 s, 72°C for 15 s]x40. Cq values for phage of known titer were used to generate a standard curve, and other samples’ Cq values were used to calculate phage titer in pfu/mL.