- YPD media, both broth and 2% agar plates]
- Glycerol stocks of knockout strains
- Cell 60 mm Culture Dish, containing 6 ml of agar
- Incubator set to 30°C.
- Bachur & Associates Sanata Clara, CA 95050 Model LS-100-3 UV Light Exposeure System
- Three days before the experiment, streak knockout strains onto YPD plates.
- One day before the experiment, pick a colony from each strain into 5 ml YPD broth in a disposable test-tube. Incubate on the roller drum at 30°C overnight.
- The morning of the experiment, dilute each culture to an OD600 of 0.2 in YPD broth. Return to the drum roller for between 30 and 90 minutes.
- Transfer 33 µl to a well in the Culture Dish.
- Set up the UV light Exposure System as follows:
- 400 watts
- time increments
- place in the incubator in dark for 48 hours
We chose to use the control of 700 seconds given the fact that it had cell death but a small amount of colonies present.