| Phage Resistant Mutant Isolation 噬菌体抗性突变体分离 | 点击： 作者： 来源： 时间： 2007-04-11 本站论坛
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|  | Hancock Laboratory Methods. Department of Microbiology and Immunology,
University of British Columbia, British Columbia, Canada http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=54 OBJECTIVE:
To isolate phage resistant mutants.
REAGENTS:
- overlay agar in tubes: 0.6% agar, 1% PP2
- 1% PP2 plates.
- host bacerial culture
METHODS:
Overlay Method
- Grow host bacteria overnight. Dilute 1:10.
- Add 0.1 ml phage stock solution and 0.1 ml of diluted bacterial culture into prewarmed overlay agar (temperature maintained at 45ºC).
- Swirl overlay agar and pour onto a PP2 plate. Do at least 3 plates to increase your chances of getting mutants.
- Incubate at 37ºC. for 2-3 days.
- Pick individual colonies with sterile toothpicks onto fresh PP2 plates. Streak for single colony. Incubate at 37ºC. overnight.
- Freeze mutants with DMSO.
Spreading Method
- 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.
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