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Preparation of Helper Phage - M13KO7

  1. Infect 200 mg/mL XLI-Blue at an OD600 of 0.4 with 10 mL of 100-fold serial dilutions of M13KO7 helper phage in a 37 ℃ water bath for 30 min. Add to 3 mL molten top agar (50 ) and pour onto warm LB plates. Allow to set and incubate overnight at 37 .
  2. Pick a small plaque into 5 mL of fresh SB at an OD600 of 0.4. Grow for about 2hr shaking at 37 .
  3. Add to 500 mL SB in a 2 litre flask and grow shaking at 37  for 1hr. Add kanamycin to a final concentration of 50 mg/mL. Grow overnight shaking at 37 .
  4. Spin overnight culture at 10,800 g for 15 min. Add 100 mL 5*PEG/NaCl to 400 mL supernatant and leave for 1 hr on ice.
  5. Spin 10,800 g for 30 min. Pour away PEG/NaCl.
  6. Re-suspend the pellet in 8 mL PBS and spin at 11,600 g for 10 min to remove any remaining bacterial debris (the titer of helper phage should be in the range of 1012-1013 pfu/mL).

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