OBJECTIVE: To isolate phage resistant mutants.

REAGENTS:

overlay agar in tubes: 0.6% agar, 1% PP2

1% PP2 plates.

host bacerial culture

OVERLAY METHOD

1. Grow host bacteria overnight. Dilute 1:10.
2. Add 0.1 ml phage stock solution and 0.1 ml of diluted bacterial culture into prewarmed overlay agar (temperature maintained at 45^oC).
3. Swirl overlay agar and pour onto a PP2 plate. Do at least 3 plates to increase your chances of getting mutants.
4. Incubate at 37^oC. for 2-3 days.
5. Pick individual colonies with sterile toothpicks onto fresh PP2 plates. Streak for single colony. Incubate at 37C. overnight.
6. Freeze mutants with DMSO.

SPREADING METHOD

1. Add 0.1 ml phage solution (10^-2 dilution) and 0.1 ml diluted bacterial culture onto a PP2 plate and spread plate.

NOTE: This method does not give uniform lawns as predictably as the overlay method, but you don't have to make the overlay agar.