Protein Protocols Protein Protocols

Phage Display 1067
8. Shake vigorously at 37°C until mid-log phase (OD 600 approx 0.5).
9. Set up for titering while cells are growing.
10. Melt Agarose Top in microwave oven (3 min on high setting).
11. Add 3 mL of Agarose Top into sterile culture tubes (one tube/dilution) in a 45°C water bath.
12. Prewarm LB plates (one plate/dilution) at 37°C until needed.
13. Prepare 10-fold serial dilutions (10 8 –10 11 for amplified eluates and phage libraries and
10 1 –10 4 for unamplified eluates).
14. Dispense 200 µL of culture at mid-log phase into microcentrifuge tubes (one tube/dilution).
15. Add 10 µL of each dilution to microcentrifuge tubes containing culture.
16. Mix on a vortex mixer.
17. Incubate at room temperature for 5 min.
18. Add infected cells to preequilibrated culture tubes containing Agarose Top.
19. Vortex-mix rapidly.
20. Pour onto prewarmed LB plate.
21. Spread Agarose Top evenly to cover surface of plate.
22. Cool plates at room temperature for 5 min with cover slightly off.
23. Cover plates completely.
24. Incubate inverted plates overnight at 37°C.
25. Count plates having approx 10 2 plaques. Multiply number of plaques with dilution factor
to determine the pfu/10 µL.
3.3.2. Phage Amplification
1. Prepare an overnight culture by inoculating a single colony of XL1-Blue in LB broth
containing 12.5 µg of Tetracycline (20 mL) in a 250-mL shake flask.
2. Incubate at 37°C with vigorous shaking.
3. Add eluate to a 1100 dilution of the overnight culture of XL1-Blue in LB broth (20 mL)
containing Tetracycline in another shake flask.
4. Incubate culture with vigorous shaking for 4.5 h at 37°C.
5. Spin culture in a centrifuge tube for 10 min at 12,000g at 4°C.
6. Recentrifuge supernatant in another centrifuge tube.
7. Pipet supernatant into another centrifuge tube.
8. Add 1/6 vol of PEG to precipitate phage at 4°C overnight.
9. Sediment precipitated phage by centrifugation at 12,000g at 4°C for 15 min.
10. Discard supernatant.
11. Recentrifuge for 1 min.
12. Remove residual supernatant.
13. Suspend pellet in 1 mL of Tris.
14. Place suspension in a microcentrifuge tube.
15. Centrifuge at high speed in a microcentrifuge for 5 min at 4°C.
16. Reprecipitate phage with 1/6 vol of PEG in a new tube.
17. Incubate on ice for 60 min.
18. Sediment phage in microcentrifuge at high speed for 10 min at 4°C.
19. Remove supernatant.
20. Recentrifuge.
21. Discard any residual supernatant.
22. Suspend pellet in 200 µL of Tris buffer containing 0.02% NaN3. 23. Sediment insoluble matter by centrifugation for 1 min at high speed in a microcentrifuge.
24. Transfer amplified eluate in supernatant to new tube.
25. Titer amplified eluate (see Subheading 3.3.1.).