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.

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 1. 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 3 79.45 20.55 4 61.45 38.55 5 48.5 51.5 6 38.86 63.15 7 17.65 82.35

== Calibration Experiment protocol ==

== Final Experiment protocol ==