Difference between revisions of "UW-Stout/Co Culture SP21"
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==Materials== | ==Materials== | ||
| − | * | + | * 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]] | ||
| − | * | + | * [https://www.thomassci.com/Molecular-Diagnostics/Extraction/Deep-Well-Plates/_/labForce-96-Well-Deep-Round-and-Square-Plates?q=96%20Deep%20Well%20Plates Thomas Scientific square,deep 96 well, round bottom micro assay plate] |
==Equipment== | ==Equipment== | ||
Revision as of 13:50, 13 April 2021
This page is under construction
Materials
- Double strength synthetic-defined media (liquid)
- Single-strength synthetic-defined media (liquid)
- Phosphate buffered saline, sterile.
- Glycerol stocks of knockout strains
- Thomas Scientific square,deep 96 well, round bottom micro assay plate
Equipment
- Shaking incubator set to 600 rpm and 30°C
- flow cytometer
- type 1000, 200, and 20 micro pipettors
- Hemocytometer
Protocol
- Create concentration of 100 cells per micro liter for both the knockout and control fluorescent strain with the Hemocytometer within 1 mL of double strength synthetic media.
- Place contents into a 2 mL culture within a deep well microassay plate.
- Repeat steps 1 and 2 for each additional yeast strain, assigning each a unique well within the microassay plate.
- Incubate the plate within the incubator shaker for 48 hours at 30 degrees Celsius, shaking at 600 rpm.
- From each well, take a 5 micro liter sample and mix it with 500 micro liters of phosphate buffered saline to measure on the flowcytometer.
