UW-Stout/Nitrogen starvation FA19

Materials:

Materials Nitrogen + Nitrogen - Histidine (x100) 1 ml 1ml Leucine (x100) 1ml 1ml Uracil (x100) 1ml 1ml CSM 65 mg 65mg YBN 171mg 171mg Glucose (40% w/v) 5ml 5ml Ammonium Sulfate 0 500mg Water 92ml 92ml

Equipment: Molecular Devices SpectraMax Plus 384 Microplate Reader

Procedure:

Part 1

Label two seperate 250ml glass media jars with Nitrogen + and Nitrogen - 

In Nitrogen + media jar mix in correct portions from above materials list Histidine (x100) Leucine (x100) Uracil (x100) CSM YBN Water In Nitrogen - Media jar mix in correct portions form above materials list Histidine (x100) Leucine (x100) Uracil (x100) CSM YBN Ammonium Sulfate Water

Mix with stirring for 10-15 minutes

Place both glass media jars in plastic bin with autoclave tape secured around a loosened lid, then proceed to autoclave at 121°C for 15 minute With autoclave gloves on retrieve the solutions When both media jars are cool, mix in 5 ml of glucose to both Nitrogen + and Nitrogen - solutions

Part 2 Gather all yeast culture media In Biosafety cabinet pipet 1ml of each culture twice into separate microcentrifuge tubes Centrifuge tubes for 30 sec at 5000 rcf In biosafety cabinet aspirate supernatant into beaker of bleach. Label tubes with 1 nitrogen - and 1 nitrogen + for each yeast culture Add 95ml of solution corresponding to the label Vortex solutions Pipet all of the solution into wells on the microplate