UW-Stout/Sodium Hydroxide SP21
Contents
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
- Vortex the yeast culture briefly to resuspend the yeast cells.
- 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.
- 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
- Add 50uL of yeast culture to each well.
- Add 50 uL of sterile water to each well.
- 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.
- 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.
- Vortex the yeast culture briefly to resuspend the yeast cells.
- 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
- 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
- Transfer the assay plate to the reader and read for 24 hours.
- Note: Repeat the experiment a few times to account for natural variation.