UW-Stout/Growth Curve SP21

Materials

• Corning COSTAR 96-well clear flat-bottom assay plate
• Falcon round-bottom polypropylene tubes
• YPD media, in 2% agar plates
• Double-strength synthetic-defined media (liquid)
• Phosphate buffered saline, sterile.
• Glycerol stocks of knockout strains

Equipment

• Incubator set to 30°C.
• New Brunswick TC-7 Tissue Culture Roller Drum
• UV/vis spectrophotometer and cuvettes
• Molecular Devices SpectraMax Plus 384 Microplate Reader

Protocol

1. Three days before the experiment, streak knockout strains onto YPD plates. For repeat experiments, store these plates in the refrigerator after they've grown up.
2. The evening 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, at least two hours before starting:
   1. Centrifuge the overnight cultures for 2 minutes at 1000 xg in a swinging-bucket centrifuge.
   2. Aspirate the YPD media.
   3. Resuspend in 5 ml PBS.
   4. Measure the OD 600 of each tube. (Dilute 1:10 in PBS if necessary to get the OD600 into the linear range of your instrument.)
   5. Aliquot 5 ml of double-strength synthetic-defined media into disposable test-tubes.
   6. Add cells-in-PBS to a final OD600 of 0.2.
      • If the required volume is less than 500 ul, it's fine to just add to the 2x SD media.
      • Otherwise, aliquot out the required amount to a new tube, centrifuge as above, then resuspend in media.
4. Transfer 50 µl of yeast culture and 50 µl of sterile water 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 18 hours.