Difference between revisions of "UW-Stout/Growth Curve SP21"

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(Created page with "==Materials== * [https://www.corning.com/worldwide/en/products/life-sciences/keymatch/3370.html Corning COSTAR 96-well clear flat-bottom assay plate] * [https://www.fishersci....")
 
(Materials)
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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)  
 +
* Single-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)
 
* [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 13:43, 13 April 2021

Materials

Equipment

Protocol

  1. Three days before the experiment, streak knockout strains onto YPD plates.
  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 24 hours.