UW-Stout/Ammonium Sulfate SP21
Contents
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
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 Protocol
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Knock Out Strain Protocol
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