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.

Knock Out Strain Protocol

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