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Packaging of phages

(1) Prepare M13KO7 helper phage

  1. Prepare helper phage by infecting log-phase TG1 bacterial cells with M13K07 phage at different dilutions for 30 min at 37°C and plating in top agar onto 2TY plates.
  2. Inoculate a small plaque in 3 mL liquid 2TY medium. Add 30 μL overnight culture of TG1 and grow for 2 h at 37°C.
  3. Dilute the culture in 1 L 2TY medium and grow for 1 h. Add Kanamycin to 50μg/mL and grow for 16 h at 37°C.
  4. Remove cells by centrifugation (10 min at 5000g) and precipitate phage from the supernatant by addition of 0.25 vol of phage precipitant. After 30 min incubation on ice, collect the phage particles by centrifugation during 10 min at 5000g. Resuspend the pellet in 5 mL PBS and sterilize through a 0.22-μm filter.
  5. Titrate the helper phage by determining the number of plaque-forming units (pfu) on 2TY plates with top-agar layers containing 100μL TG1 (saturated culture) and dilutions of phage. Dilute the phage stock solution to 1 × 1013 pfu/mL and store in small aliquots at –20°C.

(2) Prepare Library Phages

  1. Inoculate 500 mL 2TY-G with the library glycerol stock and incubate at 37°C with shaking at 250 rpm until the optical density at 600 nm reaches 0.8–0.9.
  2. Add M13KO7 helper phage to a final concentration of 5 ´109 pfu/mL, and incubate for 30 min at 37°C without shaking, then for 30 min with gentle shaking (200 rpm), to allow phage infection.
  3. Recover the cells by centrifugation at 2200g for 15 min and re-suspend the pellet in the same volume of 2TY-AK. Incubate overnight at 30°C with rapid shaking (300 rpm).
  4. Pellet the cells by centrifugation at 7000g for 15 min at 4°C and recover the supernatant containing the phage into pre-chilled 1-L bottles.
  5. Add 0.3 vol of phage precipitant. Mix gently and allow the phage to precipitate for 1 h on ice.
  6. Pellet the phage by centrifuging twice at 7000g for 15 min in the same bottle at 4°C. Remove as much of the supernatant as possible and re-suspend the pellet in 8 mL PBS.
  7. Re-centrifuge the phage in smaller tubes at 12,000g for 10 min and recover the phage via the supernatant. Ensure that any bacterial pellet that appears is left undisturbed.
  8. Finally, titer phage stocks by infecting TG1 cells with dilutions of phage stock, plating to 2TY-AG, incubation, and enumeration of the numbers of ampicillin resistant colonies that appear. The phage can then be stored in aliquots at 4°C for long periods, ready for screening.

(3) Quality control PCR of end Fab library

Using two primers [VH and VL] located at SP1 and SP2, QC PCR was conducted.

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