UW-Stout/Ultraviolet Light FA22
Contents
Methods (Calibration Experiment)
Materials
- 60mm plates with YPD media
- Yeast to be tested (suspended in media)
- Phosphate buffered saline (1X, pH 7.4)
- Glass beads for plating
Equipment
- Spectrophotometer
- Model LS-100-3 UV Exposure System
Procedure
Sample Prep
- Use a spectrophotometer to measure the optical density of the stock yeast at 600 nm.
- Dilute a sample to 25 µL at a concentration of 1 cell/µL using phosphate-buffered saline. Vortex to mix.
- Pour 4-8 beads on the plate.
- Pipette 25 µL of dilute cells onto the plate.
- Shake to spread the sample on a petri dish.
- Dispose of glass beads.
UV Exposure
- Input sample plates into the UV exposure system at 400 watts for 10, 20, 50, 100, 200, 300, 400, 500, and 600 seconds.
- Incubate plates at 30 °C in the dark for 24-48 hours.
- Measure the number of colonies.
Calibration Experiment Data
The number of colonies on each plate:
- 10 seconds - 18 colonies
- 20 seconds - 19 colonies
- 50 seconds - 12 colonies
- 100 seconds - 11 colonies
- 200 seconds - 12 colonies
- 300 seconds - 12 colonies
- 400 seconds - 6 colonies
- 500 seconds - 1 colony
- 600 seconds - 0 colonies
Calibration Experiment Interpretation
Based on the data provided by the calibration experiment, the knock-out experiment protocol will utilize a 400-second exposure period. This period was shown to be a middle point in the number of colonies killed off.
Knock-out experiment Protocol
Materials
- 60mm plates with YPD media
- Yeast to be tested (suspended in media)
- Phosphate buffered saline (1X, pH 7.4)
- Glass beads for plating
Equipment
- Spectrophotometer
- UV exposure system
Procedure
Sample Prep
- Use a spectrophotometer to measure the optical density of the stock yeast at 600 nm.
- Dilute a sample to 25 µL at a concentration of 4 cells/µL using phosphate-buffered saline. Vortex to mix.
- Pour 4-8 beads on the plate.
- Pipette 25 µL of dilute cells onto the plate.
- Shake to spread the sample on a petri dish.
- Dispose of glass beads.
UV Exposure
- Input sample plates into the UV exposure system at 400 watts for 400 seconds.
- Incubate plates at 30 °C in the dark for 24-48 hours.
- Measure the number of colonies.
