UW-Stout/Sodium Hydroxide 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
• Sodium Hydroxide, at a concentration of 0.15 M
• P2, P20, P200 micropipettors

Equipment

• Molecular Devices SpectraMax Plus 384 Microplate Reader
• Safety Gloves
• Safety Glasses
• Fume Hood
• Sterile Water

Precautions

Sodium Hydroxide is very corrosive. It can cause severe eye irritation, skin irritation, and can burn your nose. A fume hood, safety glasses and goggles, and a lab coat will be required to handle this substance safely.

Calibration Protocol

1. Vortex the yeast culture briefly to resuspend the yeast cells.
2. Various molarities of 1 mM, 5 mM,20 mM,100 mM, 150 mM,200 mM,250 mM. A total volume will always be 1 ul of Sodium Hydroxide solution.
3. Set up 8 wells as follows:
   • Well 1: 50uL Wild-Type, 50uL sterile water
   • Well 2: 50uL Wild-Type, 1mM NaOH, 49uL sterile water
   • Well 3: 50uL Wild-Type, 5mM NaOH, 49uL sterile water
   • Well 4: 50uL Wild-Type, 20mM NaOH, 49uL sterile water
   • Well 5: 50uL Wild-Type, 100mM NaOH, 49uL sterile water
   • Well 6: 50uL Wild-Type, 150 mM NaOH, 49uL sterile water
   • Well 7: 50uL Wild-Type, 200mM NaOH, 49uL sterile water
   • Well 8: 50uL Wild-Type, 200mM NaOH, 49uL of sterile water
4. Add 50uL of yeast culture to each well.
5. Add 50 uL of sterile water to each well.
6. 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.
7. Transfer the assay plate to the reader and read for 24 hours.

Raw Data from the Calibration Protocol

From this set of curves, the optimal molarity to use would be a concentration .15 M Sodium Hydroxide

Protocol

Use the data from the calibration protocol for the optimal molarity of .15 M of Sodium Hydroxide.

1. Vortex the yeast culture briefly to resuspend the yeast cells.
2. Set up eight wells aliquoting 49uL of sterile water, 1uL of sodium hydroxide, and 50uL of yeast culture. Use the seventh and eighth well for the wild-type yeast strain.
   • The molarity of the sodium hydroxide will be .15 M
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.
   • Note: Repeat the experiment a few times to account for natural variation.