UW Stout/Nystatin SP22

Pilot Protocol

Purpose: Using a stock solution of nystatin, DMF, and sterile water to treat wild yeast cells. The results of this will be used to decide on a concentration level to apply to different yeast strains.

Materials: 1. Nystatin powder 2. Dimethylformamide (DMF) 3. Sterile water 4. 1.7ml Microcenterfuge tubes 5. Weighing boat 6. Corning COSTAR 96-well clear flat-bottom assay plate 7. Glycerol stock of BY4735 strain as prepared in steps 1-4 of the basic growth curve protocol

Required Equipment: 1. Vortex 2. Scale 3. Molecular Devices SpectraMax Plus 384 Microplate Reader set as outlined in the basic growth curve protocol

Protocol For Pilot Experiment Steps to make stock solution: • weigh out 40mg of Nystatin power • Add nystatin to tube • Measure 10ml DMF • Add DMF to tube • Measure sterile water • Mix until no powder is present

Dilutions for pilot experiment Steps to make dilutions: 1. Mix sterile water with the stock solution, use table below for the amount of each to use 2. Repeat until all concentrations are complete 3. Add 50ul of the diluted product with 50ul of wild yeast

Dilution table: Desired Concentrations 1. 0ug/ml 0ug/ul 2. 0.5ug/ml or 0.0005ug/ul 3. 0.75ug/ml 0.00075ug/ul 4. 1.0ug/ml or 0.001ug/ul 5. 1.25ug/ml or 0.00125ug/ul 6. 1.5ug/ml or 0.0015ug/ul 7. 1.75ug/ml or 0.00175ug/ul 8. 2.0ug/ml or 0.002ug/ul

Trials 1 and 2 protocol Purpose: Create a nystatin treatment that is able to slow down the growth of different yeast strains.

Materials: 1.Nystatin powder 2.Dimethylformamide (DMF) 3.Sterile water 4.1.7ml Microcenterfuge tubes 5.Corning COSTAR 96-well clear flat-bottom assay plate 6.Glycerol stock of BY4735 strain as prepared in steps 1-4 of the basic growth curve protocol

Required Equipment: 1.Vortex 2. Micropipettes

Protocol for trials 1 and 2: 1.Measure out stock solution and place it in 1.7 tube 2.Measure out and add water to the tube 3.Vortex the mixture 4.Apply 50ul of product to each yeast strain

Dilutions: