UW-Stout/Hydroxyurea SP21

Materials

• Corning COSTAR 96-well clear flat-bottom assay plate
• Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
• Hydroxyurea (HU)
• P20, P100 and P200 micropipettors

Equipment

• Molecular Devices SpectraMax Plus 384 Microplate Reader

Precautions

Lab equipment (gloves, eyewear, lab coat) worn at all times. Hydroxyurea is dangerous when exposed to skin, but it is OK to work with.

Calibration Protocol

1. Vortex the yeast culture briefly to resuspend the yeast cells. The starting optimal density should be 0.2.
2. Create a working stock of 1 mL @ 1 M [Should be 0.076g of HU w/ 1 mL of water] Measure using the analytical balance
3. Create multiple stock solutions: 10mM, 20mM, 50mM, 100mM, 200mM. With distilled H2O each with a final volume of 1000 uL.
4. Prepare solutions wells like shown:
   • 0 mM – 0g of HU
   • 10 mM – 10 ul of 1M stock + 990ul H2O
   • 20 mM – 20U ul of 1M stock + 980 ul H2O
   • 50 mM – 50 ul of 1M stock + 950 ul H2O
   • 100 mM – 100 ul of 1M stock + 900 ul H2O
   • 200 mM – 200 ul of 1M stock + 800 ul H2O
5. Set up 6 wells as shown: Transfer 50ul of each stock solution to each well.
   • Well 1: 0ul HU, 50ul sterile water (final concentration 0mM)
   • Well 2: 50uL HU, 0uL sterile water (final concentration 10mM)
   • Well 3: 50ul HU, 0ul sterile water (final concentration 20mM)
   • Well 4: 50ul HU, 0ul sterile water (final concentration 50mM)
   • Well 5: 50ul HU, 0ul sterile water (final concentration 100mM)
   • Well 6: 50ul HU, 0ul sterile water (final concentration 200mM)
6. Add 50uL of yeast culture to each well,for a final volume of 100 uL.
7. To measure cell growth over a period of 24 hours, set up the plate reader as follows:
   • Temperature: 30°C
   • Mode: Kinetic
   • Wavelength: 600 nm
   • Interval: 5 minutes
   • Total run time: 24 hours
   • Shake before reading: 30 seconds.
8. Transfer the assay plate to the reader and read for 24 hours.

Raw Data from the Calibration Protocol

Concentrations in the legend are final concentrations of Hydroxyurea. In this set of curves, a concentration of 100mM is the optimal concentration of the stressor to test the effect of Hydroxyurea on yeast cells because it applies stress on the yeast to inhibit its growth, but will not prevent growth to the point of cell death.

Protocol

Use the data from the calibration protocol for the optimal concentration of Hydroxyurea.

1. Vortex the yeast culture briefly to resuspend the yeast cells.
2. Set up six wells 50uL of the stock hydroxyurea and 50uL of yeast culture.
   • The final concentration of hydroxyurea is 100mM.
3. To measure cell growth over a period of 24 hours, set up the plate reader as follows:
   • Temperature: 30°C
   • Mode: Kinetic
   • Wavelength: 600 nm
   • Interval: 5 minutes
   • Total run time: 24 hours
   • Shake before reading: 30 seconds
4. Transfer the assay plate to the reader and read for 24 hours.
   • Repeat the experiment a few times to allow for variation.