UW-Stout/pH Acid SP22

Introduction

Our protocol is designed to determine if the knocked-out gene affects or does not affect the growth of our yeasts in environments of pH 7. The pH of 7 was predetermined in a pilot experiment to be a pH that would stress the cell’s growth without killing all the cells. A citric acid monohydrate buffer was used to maintain a pH of 7 in the growth environments throughout the entire growth process.

Safety

Inhalation of disodium phosphate can lead to a cough, shortness of breath, and a sore throat. Getting disodium phosphate on your skin can lead to a rash and swelling. Getting it into your eyes can lead to redness and swelling also. Inhalation of citric acid monohydrate can lead to erosion of the teeth2. A fume hood, safety glasses and goggles, gloves and a lab coat will be required to handle this substance safely.

Materials/Equipment

• Citric Acid Monohydrate
• Disodium Phosphate
• De-ionized Water
• Micropipette
• 15ml tube (3x)
• 96-well clear flat-bottom assay plate
• Micropipette tips
• Gloves
• Incubator set to 30 C
• Scale
• Sterile cabinet
• pH test strips
• Yeast strains

Procedure

Preparing Buffer Stock Solutions (0.1M citric acid, 0.2M disodium phosphate)

1. To obtain 0.1M of citric acid monohydrate, combine .21g of in 10ml of de-ionized water
2. To obtain 0.2M of disodium phosphate, combine .28g in 10 ml de-ionized water

Preparing Buffer Solution To create a buffer solution at a specific pH, you must combine x ml of citric acid monohydrate with y ml of disodium phosphate and shake well pH x ml 0.1M-Citric Acid y ml 0.2M-Na2HPO4

Citric Acid Monohydrate-Disodium Phosphate Buffer Calibration Experiment

In this experiment, the pH of Citric Acid Monohydrate-Disodium Phosphate Buffer needs to have an effect on the yeast cells that is not too aggressive towards the yeast cells but affects them to show a difference.

Plating

1. Determine the “sweet spot” solution
2. Vortex yeast cells to resuspend the yeast cells briefly
3. Various pHs of 3 pH, 4 pH,5 pH,6 pH, and 7 pH,10 pH, 10.2 pH were tested in this experiment. A total volume will always be 50 ul of the Citric Acid Monohydrate-Disodium Phosphate buffer in this experiment. The table shows the pH of Citric Acid Monohydrate-Disodium Phosphate buffer in each well. All plates will be a total volume of 100 ul.

• remember OD600 is the "optical density" of the yeast

Final Experiment Protocol

Addition of Buffer to Yeast

1. Vortex wild type strain and knock out strains
2. Add 50ul of each knock out strain to own each well
3. Add 50ul of pH buffer to each subsequent well

Preparing Plate Reader

• Temperature: at 30 degrees Celsius
• Mode: Kinetic
• Read: 600 n
• Intervals: 5 minutes
• Total run time: 24 hours
• Shake before read: 30 seconds
• Transfer the assay plate to the reader and run for 24 hours

Results

Interpretation and Doubling Time