1. Centrifuge cells at 1000g for 15 min.

2. Transfer supernatant into another centrifuge tube and centrifuge at 10,000g for 10 min.

3. Transfer supernatant into a Vivaspin 15 (Vivascience) unit. Pour 15 mL of supernatant into the unit. Centrifuge at 800g for 15 min. Add the remaining supernatant and centrifuge another 15 min at 800g. Collect the concentrate (approx 0.5 mL) and add sterile TBS to 1 mL.

4. Transfer the 1 mL of concentrate to 150 |L of of PEG/NaCl in a second Eppendorf tube. Mix by inverting 100X. Leave on ice for at least 1 h.

5. Microfuge for 10 min at max speed.

6. Remove supernatant, recentrifuge briefly, and remove supernatant.

7. Resuspend pellet in 200 |L of TBS/azide.

8. Use 100-200 |L of this purified phage and 400 |L of TBST for next panning.

This is now ready for the second round of biopanning, so that d 3 now becomes d 2 again (see Note 5).

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