UW-Stout/Growth Curve

Materials

• Corning COSTAR 96-well clear flat-bottom assay plate
• Falcon round-bottom polypropylene tubes
• YPD media, both broth and 2% agar plates
• Glycerol stocks of [UW-Stout/Knockout_Protocol|knockout strains]

Equipment

• Standing incubator set to 30°C.
• New Brunswick TC-7 Tissue Culture Roller Drum
• Molecular Devices SpectraMax Plus 384 Microplate Reader

Protocol

1. Three days before the experiment, streak knockout strains onto YPD plates.
2. One day before the experiment, pick a colony from each strain into 5 ml YPD broth in a disposable test-tube. Incubate on the roller drum at 30°C overnight.
3. The morning of the experiment, dilute each culture to an OD600 of 0.2 in YPD broth. Return to the drum roller for between 30 and 90 minutes.
4. Transfer 100 µl to a well in the assay plate.
5. Set up the plate reader as follows:
   • Temperature: 30°C
   • Mode: Kinetic
   • Read: 600 nm
   • Interval: 5 minutes
   • Total run time: 24 hours
   • Shake before read: 30 seconds
6. Transfer the assay plate to the reader and run for 24 hours.