Difference between revisions of "Team:Paris Bettencourt/Notebook/Protocols"

Notebook

Protocols

Bibliography

Phage Display Protocols

Phage Titering

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.

1. Inoculate 10mL of LB with ER2738 from a plate and incubate with shaking ~3-4 hours (mid-log phase, OD600 ~ 0.5).
2. 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°C.
3. Pre-warm, for at least one hour, one LB/IPTG/Xgal plate per expected dilu¬tion at 37°C until ready for use.
4. Prepare 10 to 103 -fold serial dilutions of phage in LB; 1 ml final volumes are convenient. Suggested dilution ranges: for amplified phage culture supernatants, 10^8 – 10^11; for unamplified panning eluates, 10^1–10^4. Use aerosol-resistant pipette tips to prevent cross-contamination, and use a fresh pipette tip for each dilution.
5. When the culture in Step 1 reaches mid-log phase, dispense 200 μl into microfuge tubes, one for each phage dilution.
6. To carry out infection, add 10 μl of each phage dilution to each tube, vortex quickly, and incubate at room temperature for 1–5 minutes.
7. Transfer the infected cells one infection at a time to culture tubes containing 45°C Top Agar. Vortex briefly and IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate (200µM IPTG, 30µg/mL X-gal). Gently tilt and rotate plate to spread top agar evenly.
8. Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C.
9. 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 10µL. Multiply by 100 to get the pfu/mL.

Panning procedure

DAY 0

• Streak ER2738 from glycerol stock on LB+Tet plate.
• Incubate overnight at 4°C the piece of fabric/thread with 1mL of blocking buffer. Alternatively, after inoculation of the titering culture incubate the piece of fabric/thread at least 1 hour at 4°C on DAY 1.

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5