UW-Stout/Bases SP23

Introduction

The goal of the experiment is to find a pH level of ammonium hydroxide and ammonium chloride buffer that upsets our yeast but does not kill them. The pH levels range from 8 to 10.5 increasing in .5 increments and then recording the effects on the growth at each level.

Materials

• Ammonium Chloride
• Ammonium Hydroxide
• Corning COSTAR 96-well clear flat-bottom assay plate
• microcentrifuge tubes
• micropipette and corresponding tips
  • p20
  • p100
  • p1000
• pH reader
• wild-type yeast in 2x synthesis complete media at an OD600 of 0.1-0.2
• 15 mL conical tubes (x6)
• 20 mL sterile water

Procedure

Make basic buffers using the ammonium chloride and ammonium hydroxide

1. add small increments of the ammonium hydroxide into the ammonium chloride while using the pH reader until you reach pH level of 10.5
2. pour buffer into a labeled conical tube
3. repeat step 1 and 2 until you have made each pH level

• 8 8.5 9 9.5 10 10.5
• once buffers are made they can be stored at room temperature

this portion of the experiment is done in a sterile environment like a biosafety cabinet MAKE SURE TO VORTEX YEAST CELLS BEFORE ADDING TO MICROCENTRIFUGE TUBES

1. in a labeled microcentrifuge tube add

• 50 ul yeast culture
• 40 ul sterile water
• 10 ul of pH level 8 buffer

1. repeat step above using each pH level
2. vortex each tube
3. pipette 100ul of each concentration into a well

• make sure to record which pH level is in each well

1. set up plate reader

• temperature 30 degrees Celsius
• mode kinetic
• wavelength 600 nm
• interval 5 minutes
• total run time 24 hours
• shake before read 30 seconds

1. transfer assay plate into reader and read for 24 hours