Difference between revisions of "UW-Stout/Growth Curve SP21"
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* [https://en.wikipedia.org/wiki/YEPD YPD media], in 2% agar plates | * [https://en.wikipedia.org/wiki/YEPD YPD media], in 2% agar plates | ||
* Double-strength [https://sunrisescience.com/shop/growth-media/media-for-yeast/sd-complete-ynbnitrogen-glucose-csm-supplement-mix/sd-broth/sd-powder-10-x-0-5-liter-pouches/ synthetic-defined media] (liquid) | * Double-strength [https://sunrisescience.com/shop/growth-media/media-for-yeast/sd-complete-ynbnitrogen-glucose-csm-supplement-mix/sd-broth/sd-powder-10-x-0-5-liter-pouches/ synthetic-defined media] (liquid) | ||
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* [https://en.wikipedia.org/wiki/Phosphate-buffered_saline Phosphate buffered saline], sterile. | * [https://en.wikipedia.org/wiki/Phosphate-buffered_saline Phosphate buffered saline], sterile. | ||
* Glycerol stocks of [[UW-Stout/Knockout_Protocol|knockout strains]] | * Glycerol stocks of [[UW-Stout/Knockout_Protocol|knockout strains]] |
Revision as of 13:48, 13 April 2021
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
- Three days before the experiment, streak knockout strains onto YPD plates.
- 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.
- The morning of the experiment, at least two hours before starting:
- Centrifuge the overnight cultures for 2 minutes at 1000 xg in a swinging-bucket centrifuge.
- Aspirate the YPD media.
- Resuspend in 5 ml PBS.
- 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.)
- Aliquot 5 ml of double-strength synthetic-defined media into disposable test-tubes.
- 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.
- 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.