Day culture. Inoculate 5–10 ml of LB with E. coli K12 ER2738 (NEB #E4104S) from a plate and incubate with
shaking 4–8 hrs until mid-log phase, OD600 ~ 0.5.
Prepare top agar and LB/IPTG/Xgal plates. While cells
are growing, melt Top Agar in microwave and dispense 3 ml into sterile
culture tubes, one per expected phage dilution. Maintain tubes at 45–50°C.
Pre-warm, for at least 30 minutes, one LB/IPTG/Xgal plate per expected
dilution at 37°C until ready for use.
Phage Dilutions. Prepare 10 to 103-fold
serial dilutions of phage in LB; 1 ml final volumes are convenient.
Suggested dilution ranges: for amplified-PEG/NaCl concentrated phage
culture supernatants, 108–1011; for unamplified
panning eluates, 101–104. Use aerosol- resistant
pipette tips to prevent cross-contamination and use a fresh pipette tip for
Pre-plating Infections. When the culture in Step 1 is
turbid, dispense 200 μl into microfuge tubes, one for each phage dilution.
To carry out infection, add 10 μl of each phage dilution to each tube of
cells, vortex quickly, and incubate at room temperature for 1–5 minutes.
Plating. Transfer the infected cells one infection at a
time to culture tubes containing warm Top Agar. Vortex briefly and
IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate while
maintaining sterile conditions. Gently tilt and rotate plate to spread top
agar evenly. Allow the plates to cool for 5 minutes, invert, and incubate
overnight at 37°C.
Count plaques the next day. Count blue plaques on
plates. Multiply each number by the dilution factor for that plate to
calculate phage titer in plaque forming units (pfu) per 10 μl. For example,
if there are 91 pfu on a 109 dilution plate, the titer result
would be 9.1 x 109 pfu/μl for the stock.