The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e., cells are in considerable excess). For this reason, it is recommended that phage stocks be titered by diluting prior to infection, rather than by diluting cells infected at a high MOI. Plating at low MOI will also ensure that each plaque contains only one DNA sequence.
- Inoculate 5–10 ml of LB with a swatch of ER2738* cells grown on an LB/Tet plate and incubate with shaking 4–8 hrs to mid-log phase (OD600 ~ 0.5).
- 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 one hour, one LB/IPTG/Xgal plate per expected dilution at 37°C until ready for use.
- Prepare dilutions using 10 to 103-fold serial dilutions of phage in LB; 1 ml final volumes are convenient. Suggested dilution ranges: for amplified phage stocks and infected 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 each dilution.
- When the culture in Step 1 reaches mid-log phase, 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, mix briefly, and incubate at room temperature for 1–5 minutes.
- 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. 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 on plates that have approximately 100 plaques. Multiply each number by the dilution factor for that plate to get phage titer in plaque forming units (pfu) per volume, e.g. 78 plaques from 10 ul of 108 dilution in the infection indicates a stock titer of 7.8 x 108 pfu/ul.
*Strain notes: There are many suitable E. coli
strains although ER2738 or NEB Turbo are our preference. The strain must have an F’. Ph.D.™ libraries should be used in glnV
Per liter: 10g Bacto-Tryptone, 5 g yeast extract, 5g NaCl
Mix 1.25 g isopropyl-β-D-thioglactoside (IPTG) and 1 g 5-bromo-4-chloro-3-indoyl- β-D-glactoside (Xgal) in 25 ml dimethyl formamide (DMF). Solution can be stored at -20 °C.
LB/ IPTG/Xgal Plates
1 liter of LB medium + 15 g/l agar. Autoclave, cool to <70 °C, add 1 ml IPTG/Xgal stock per lier and pour. Store at 4°C in the dark.
Per liter: components of LB Medium + 7 g Bacto-Agar(or electrophoresis grade agarose). Autoclave, dispense into 50 ml aliquots. Store at room temperature, melt in microwave as needed.