Phage fluorescent labeling

Pass filtered phage lysate (phages removed from bacteria after growth) once through a 100 kDa Amicon filter (in a benchtop centrifuge, not an ultracentrifuge) to exchange the media to PBS (pH 7.4). This helps get rid of molecules in the lysate that the dye might bind to. See my protocol below.

Dye prep

Only done when receiving new dye, not each time labeling occurs.

• Fresh 1 mg AZdye TFP ester 488 (or 568 or … you can choose your own based on your filter set) is dissolved into 200 uL water.
• 10 uL aliquots are made into centrifuge tubes.
• Dye is desiccated by running these samples through a speedvac, and the tubes are stored at -20 C in an airtight container with desiccant until further use.

Labeling

• A dye aliquot is taken out of the freezer and incubated at room temperature in an airtight container (e.g., a 50 mL centrifuge tube) with desiccant.
• Phage lysate (100-500 uL) is dissolved in PBS to a final volume of 4 mL, filtered using 100 kDa Amicon filters, and resuspended in 100 uL PBS.
• 100 uL of the phage lysate is transferred to the tube with the dye aliquot and pipette-mixed.
• The tube is incubated with gentle shaking at room temperature for 2-3 hours. Optional: overnight incubation at 4 C.
• Excess dye is washed (through Amicon filters) 4X with PBS.