UW-Stout/Ammonium Sulfate SP21

Materials

• Yeast Media Ingredients
  • Yeast nitrogen base (YNB)
  • CSM His-Leu-Ura
  • Water
  • 40% w/v Glucose Solution
• P1000/P200/P20/P10 Micropipettes with corresponding tips
• 1.5 ml Microcentrifuge tubes
• 96 well plate
• Sterile Water
• Media glassware with cap
• 50 ml beaker

Equipment

• Molecular Devices SpectraMax Plus 384 Microplate Reader

Preparation

Ammonium Sulfate Lacking Solution

First the solution with out ammonium sulfate needs to be made. Mix the following ingredients into a piece of glassware with a removable cap. The order does not matter, though it is good to start with adding the water.

• 0.342g YNB
• 0.13g CSM
• 2 ml of 100x His-Leu-Ura solutions
• 95 ml of water

Once mixed thoroughly, autoclave for 30 minutes. Once glassware is reasonably cool, add 10 ml of glucose.

Prepare the 5g/ml Ammonium Sulfate Solution and the 100x Dilution

In a 50 ml beaker mix the following

• 0.05g of Ammonium sulfate
• 10 ml of water

Prepare the 100x dilution by mixing the following in 150 ml beaker

• 1 ml of 5g/ml solution
• 99 ml of water

Will only need a filled microcentrifuge tube of each so transfer the adequate amount for easier handling of solutions.

Switching Yeast from Ammonium Sulfate Containing Media to Lacking Media

To separate the yeast cells from media, follow these steps. NOTE: When handling the live yeast cells operate under the hood!

• Vortex stock solution of cells.
• Transfer 1 ml of cells into microcentrifuge tube.
• Centrifuge at 1000 rcf for 2 minutes.
• Pipette off the supernatant leaving the pellet (the cells).
• Apply a PBS solution rinse and filter out PBS
• Transfer 1 ml of the ammonium sulfate lacking media prepared earlier into the microcentrifuge tube.
• Vortex to resuspend the cells.

Wild Yeast vs. Differing Treatment Plating

In your designated well column, for example #7, pipette the following amounts into each of the corresponding rows.

A: 50ul Cells, 40ul H20, 10ul Ammonium Sulfate solution

B: 50ul Cells, 46ul H20, 4ul Ammonium Sulfate solution

C: 50ul Cells, 48ul H20, 2ul Ammonium Sulfate solution

D: 50ul Cells, 49ul H20, 1ul Ammonium Sulfate solution

E: 50ul Cells, 49.6ul H20, 0.4ul Ammonium Sulfate solution

F: 50ul Cells, 49.8ul H20, 0.2ul Ammonium Sulfate solution

G: 50ul Cells, 49ul H20, 1ul 100x Ammonium Sulfate solution

H: 50ul Cells, 50ul H20

Place into the incubator for 24 hours and examine plate in the plate reader. Results are listed below.

Knock Out Strain Plating

This is set up for 3 treatments at once so take care when pipetting. You will have 3 designated columns, for example use #1-3, and will use a different strain in each of the rows. Order your plate as the information below shows and transfer the correct volumes.

Each Row will be using a different yeast strain. Be sure to be pipetting the correct yeast strains into the correct rows.

Column #1: Treatment 5g/ml Ammonium Sulfate

Wild Row A: 50ul Wild yeast Cells, 40ul H2O, 10ul Ammonium Sulfate

KO1 Row B: 50ul KO1 yeast Cells, 40ul H2O, 10ul Ammonium Sulfate

KO2 Row C: 50ul KO2 yeast Cells, 40ul H2O, 10ul Ammonium Sulfate

KO3 Row D: 50ul KO3 yeast Cells, 40ul H2O, 10ul Ammonium Sulfate

KO4 Row E: 50ul KO4 yeast Cells, 40ul H2O, 10ul Ammonium Sulfate

KO5 Row F: 50ul KO5 yeast Cells, 40ul H2O, 10ul Ammonium Sulfate

KO6 Row G: 50ul KO6 yeast Cells, 40ul H2O, 10ul Ammonium Sulfate

Column #2: Treatment: 0.5g/ml Ammonium Sulfate

Wild Row A: 50ul Wild yeast Cells, 49ul H2O, 1ul Ammonium Sulfate

KO1 Row B: 50ul KO1 yeast Cells, 49ul H2O, 1ul Ammonium Sulfate

KO2 Row C: 50ul KO2 yeast Cells, 49ul H2O, 1ul Ammonium Sulfate

KO3 Row D: 50ul KO3 yeast Cells, 49ul H2O, 1ul Ammonium Sulfate

KO4 Row D: 50ul KO4 yeast Cells, 49ul H2O, 1ul Ammonium Sulfate

KO5 Row F: 50ul KO5 yeast Cells, 49ul H2O, 1ul Ammonium Sulfate

KO6 Row G: 50ul KO6 yeast Cells, 49ul H2O, 1ul Ammonium Sulfate

Column #3: Treatment: Negative Control

Wild Row A: 50ul Wild yeast Cells, 50ul H2O

KO1 Row B: 50ul KO1 yeast Cells, 50ul H2O

KO2 Row C: 50ul KO2 yeast Cells, 50ul H2O

KO3 Row D: 50ul KO3 yeast Cells, 50ul H2O

KO4 Row E: 50ul KO4 yeast Cells, 50ul H2O

KO5 Row F: 50ul KO5 yeast Cells, 50ul H2O

KO6 Row G: 50ul KO6 yeast Cells, 50ul H2O

Place into the incubator for 24 hours and examine plate in the plate reader.