Magnetic Bead-based Mycobacteriophage Capture

Mycobacteriophage D29 was used as ligand for coating magnetic beads as previously descried by Foddai and Grant^{30 (link)} in their recent publication. This attachment was fortified by covalent bonds between free amines on the surface of bacteriophage and tosyl groups on Tosylactivated magnetic beads. Briefly, 10 mg Dynabeads MyOne Tosylactivated (Thermo Fisher, code 65501, Milan, Italy) were transferred into Eppendorf (2 mL), washed two times with 1 mL sodium carbonate decahydrate buffer (VWR, Milan, Italy) at concentration of 0.1 M and pH = 9.5 as binding buffer, and magnetically recovered. Then, beads were resuspended at the same binding buffer, exposed to mycobacteriophage D29 (Actiphage, UK, England) at concentration of 10⁸ pfu/mL, and incubated at 37 °C overnight rotating (10–20 rpm) continually. A day after, beads were recovered magnetically, washed two times with the binding buffer, and resuspended in Middlebrook 7H9 medium supplemented with 10% OADC and 2 mM CaCl₂. At the end, the phage-bead suspension was stored at 4 °C and only fifteen minutes prior inoculation transferred into the room temperature in order to be equilibrated with this temperature.