Quick Start Protocol PhD Phage Display Peptide Library Kit v2

  1. Streak E. coli K12 ER2738 (NEB #E4104S) onto LB/Tet solid medium. Grow overnight at 37°C.

  2. Select phage-target capture method: Prepare capture surface by coating with target and blocking, or select affinity (magnetic) beads method.

  3. Incubate 10 μl phage library (or 1011 pfu) with target (on surface or free in solution, see Step 2), 10–60 min.

  4. Wash unbound phage away with 10 x 1 ml washes of TBST.

  5. Release bound phage with low pH buffer or with a known ligand to the target.

  6. Amplify the selection’s phage eluate in 20 ml E. coli culture for 4–5 hr.

  7. Concentrate phage from culture supernatant by adding 1/5 volume of 20% PEG/2.5 M NaCl.

  8. Titer a) unamplified eluate from Step 5, for future reference, and b) enriched phage pool from Step 7, to determine volume of input for round 2 selection.

  9. Repeat steps 3–6, with 1011 pfu input of enriched phage pool each time, increasing selection or wash stringency if desired.

  10. After three rounds of selection, do not amplify elution. Instead, titer the third-round elution to obtain individual plaques for phage sequencing reactions.

  11. Post-panning analysis: Identify sequence motifs and/or carry out binding studies with selected clones.