4.3. Cesium Chloride Gradient Purification

Isopycnic centrifugation through caesium chloride (CsCl) gradients was performed, as previously described [56], with a number of modifications. A high-titer phage lysate (>1 × 10⁹ PFU/mL) was precipitated using polyethylene glycol (15% w/v PEG8000, 1 M NaCl) at 4 °C overnight and centrifuged, after which the pellet was resuspended in TMN buffer (10 mM Tris-HCl pH 7.4, 10 mM MgSO₄·7H₂O, 0.5M NaCl) and, where necessary, a chloroform phase separation step (1:1) was conducted to remove debris. The resulting phage preparation was placed onto a CsCl step gradient composed of 1.3, 1.5, and 1.7 g/mL layers and spun in a 100 Ti rotor (Beckman Coulter, Brea, CA, USA) at 200,480× g for 3 h at 4 °C. Resulting phage bands were collected and subjected to dialysis with two changes of Tris-HCl buffer (10 mM, pH 7.5) at 4 °C.