Phage Purification via PEG Precipitation

According to a previously described method (44 (link)), 500 mL of phage lysate was precipitated with 10% (wt/vol) PEG 6000 (Sigma-Aldrich) and 0.5 M NaCl and incubated overnight at 4°C with agitation. Bacterial cells were removed by centrifugation at 4,400 × g (Beckman Coulter, Aventi JE Centrifuge) for 30 min at 4°C. The pellet was resuspended in TM buffer. PEG was removed twice by adding an equal volume of chloroform, mixed by gentle inversion for 30 s, and centrifuged at 3,800 × g (Beckman, GS-15R Centrifuge) for 10 min. Then, 0.75 g of cesium chloride (CsCl) was added per mL of the aqueous phase containing phage particles (45 (link)). The solution was centrifuged at 31,000 rpm (Beckman Coulter, Optima L80 XP Ultracentrifuge) for 48 h at 10°C in a SW 41 Ti rotor. The phage band was recovered by piercing through the ultracentrifuge tube with a sterile 25-G needle (Terumo Corporation). To remove CsCl, the sample was transferred in a dialysis cassette G₂ (Thermo Fisher Scientific) and dialyzed against 1.5 L of TM buffer at 4°C overnight.