UW-Stout/Co Culture SP21
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Materials
- double strength synthetic-defined media (liquid)
- Phosphate buffered saline, sterile.
- Glycerol stocks of knockout strains
- Disposable deep well microassay plates with silicon covers
Equipment
- Shaking incubator set to 600 rpm and 30°C
- flow cytometer
- type 1000, 200, and 20 micro pipettors
- Hemocytometer
Protocol
- Create concentration of 100 cells per micro liter for both the knockout and control fluorescent strain with the Hemocytometer within 1 mL of double strength synthetic media.
- Place contents into a 2 mL culture within a deep well microassay plate.
- Repeat steps 1 and 2 for each additional yeast strain, assigning each a unique well within the microassay plate.
- Incubate the plate within the incubator shaker for 48 hours at 30 degrees Celsius, shaking at 600 rpm.
- From each well, take a 5 micro liter sample and mix it with 500 micro liters of phosphate buffered saline to measure on the flowcytometer.
