Home Protocols M13 Amplifcation Protocol for Ph.D. Phage Display

M13 Amplifcation Protocol for Ph.D. Phage Display

Protocol

  1. Grow overnight culture of F'+ E. coli (e.g. ER2738).
  2. Inoculate a 20 ml culture in a 250 ml Erlenmyer flask with 200 µL overnight E. coli culture. Add 1 µL phage suspension. Shake flask at 37°C, 250 rpm for 4 -5 hrs.
  3. Remove cells by centrifugation at 4500 g for 10 min. Transfer supernatant to a fresh tube. Repeat centrifugation.
  4. Transfer top 16 ml of supernatant to a new tube and add 4 mL of 2.5 M NaCl/20 % PEG-8000 (w/v). Briefly mix. Precipitate phage for 1 hr or overnight at 4°C.
  5. Pellet phage by centrifugation at 12000 g for 15 min. Decant supernatant. Resuspend pellet in 1 mL TBS. Transfer to an eppendorf tube. Spin briefly to remove any cell debris.
  6. Transfer supernatant to a fresh tube. Add 200 µL of 2.5 M NaCl/20% PEG-8000. Incubate on ice for 15-60 min. Spin 12000 -14000 rpm in a benchtop centrifuge for 10 min. Discard supernatant. Spin again briefly and remove remaining supernatant with pipette. Resuspend pellet in 200 µL TBS. For long-term storage at -20 °C, add 200 µL sterile glycerol.

    To scale up the above protocol, use multiple culture flasks. Alternatively, after incubating 20 ml culture for 2 hrs, add the entire culture to 1L LB. Incubate the large culture for 4 hrs, then modify the protocol to remove cells and purify phage.