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− | Materials:
| + | [[File:Yeast_Tourture_Ex_C+T1.jpg|1,700 × 2,200 |Caption]] |
− | | + | [[File:Yeast_Tourture_Ex._C+T2.jpg|1,700 x 2,200 |Caption]] |
− | Materials
| + | [[File:Nitrogen_Starvation_BY4735.jpg|463 x 599|Caption]] |
− | Nitrogen +
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− | Nitrogen -
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− | Histidine (x100)
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− | 1 ml | |
− | 1ml
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− | Leucine (x100)
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− | 1ml
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− | 1ml
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− | Uracil (x100)
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− | 1ml
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− | 1ml
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− | CSM
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− | 65 mg
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− | 65mg
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− | YBN
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− | 171mg
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− | 171mg
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− | Glucose (40% w/v)
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− | 5ml
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− | 5ml
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− | Ammonium Sulfate
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− | 0
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− | 500mg
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− | Water
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− | 92ml
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− | 92ml
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− | | |
− | Equipment:
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− | Molecular Devices SpectraMax Plus 384 Microplate Reader
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− | Procedure:
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− | Part 1
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− | Label two seperate 250ml glass media jars with Nitrogen + and Nitrogen -
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− | In Nitrogen + media jar mix in correct portions from above materials list
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− | Histidine (x100)
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− | Leucine (x100)
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− | Uracil (x100)
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− | CSM
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− | YBN
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− | Water
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− | In Nitrogen - Media jar mix in correct portions form above materials list
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− | Histidine (x100)
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− | Leucine (x100)
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− | Uracil (x100)
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− | CSM
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− | YBN
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− | Ammonium Sulfate
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− | Water
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− | Mix with stirring for 10-15 minutes
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− | 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
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− | With autoclave gloves on retrieve the solutions
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− | When both media jars are cool, mix in 5 ml of glucose to both Nitrogen + and Nitrogen - solutions
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− | Part 2
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− | Gather all yeast culture media
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− | In Biosafety cabinet pipet 1ml of each culture twice into separate microcentrifuge tubes
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− | Centrifuge tubes for 30 sec at 5000 rcf
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− | In biosafety cabinet aspirate supernatant into beaker of bleach.
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− | Label tubes with 1 nitrogen - and 1 nitrogen + for each yeast culture
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− | Add 95ml of solution corresponding to the label
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− | Vortex solutions
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− | Pipet all of the solution into wells on the microplate
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