Difference between revisions of "UW-Stout/Growth Curve"
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# Three days before the experiment, streak knockout strains onto YPD plates. | # Three days before the experiment, streak knockout strains onto YPD plates. | ||
| − | # | + | # The morning 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 for 8-10 hours. |
| − | # The morning of the experiment, dilute each culture to an OD600 of 0.2 in YPD broth. Return to the drum roller | + | # The evening before the experiment, transfer 20 ul of each strain into 5 ml of fresh YPD broth. Incubate on a roller drum at 30°C overnight. |
| − | # Transfer | + | # The morning of the experiment, dilute each culture to an OD600 of 0.1-0.2 in YPD broth. Return to the drum roller until ready to test. |
| + | # Transfer 50 µl of yeast culture and 50 µl of sterile water to a well in the assay plate. | ||
# Set up the plate reader as follows: | # Set up the plate reader as follows: | ||
#*Temperature: 30°C | #*Temperature: 30°C | ||
#*Mode: Kinetic | #*Mode: Kinetic | ||
#*Read: 600 nm | #*Read: 600 nm | ||
| − | #*Interval: 5 minutes | + | #*Interval: 5 minutes |
#*Total run time: 24 hours | #*Total run time: 24 hours | ||
#*Shake before read: 30 seconds | #*Shake before read: 30 seconds | ||
#Transfer the assay plate to the reader and run for 24 hours. | #Transfer the assay plate to the reader and run for 24 hours. | ||
Revision as of 08:30, 26 November 2019
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 knockout strains
Equipment
- Incubator set to 30°C.
- New Brunswick TC-7 Tissue Culture Roller Drum
- Molecular Devices SpectraMax Plus 384 Microplate Reader
Protocol
- Three days before the experiment, streak knockout strains onto YPD plates.
- The morning 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 for 8-10 hours.
- The evening before the experiment, transfer 20 ul of each strain into 5 ml of fresh YPD broth. Incubate on a roller drum at 30°C overnight.
- The morning of the experiment, dilute each culture to an OD600 of 0.1-0.2 in YPD broth. Return to the drum roller until ready to test.
- Transfer 50 µl of yeast culture and 50 µl of sterile water to a well in the assay plate.
- 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
- Transfer the assay plate to the reader and run for 24 hours.
