Difference between revisions of "UW-Stout/G-418"
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==Materials== | ==Materials== | ||
− | + | * [https://www.corning.com/worldwide/en/products/life-sciences/keymatch/3370.html Corning COSTAR 96-well clear flat-bottom assay plate] | |
− | + | * [https://www.invivogen.com/g418 Geneticin 100 mg/ml] | |
− | + | * Glycerol stocks of [[UW-Stout/Knockout_Protocol|knockout strains]] | |
− | + | * Water | |
====Strains of Yeast==== | ====Strains of Yeast==== | ||
BY4735 (wild type), | BY4735 (wild type), |
Revision as of 17:58, 5 May 2019
Contents
Materials
- Corning COSTAR 96-well clear flat-bottom assay plate
- Geneticin 100 mg/ml
- Glycerol stocks of knockout strains
- Water
Strains of Yeast
BY4735 (wild type), YEL035C, YCL002C, YFL064C, YGL235W, YCR100C, YJL043W, YPL068C, and YMR090W
Equipment
- Micropipettes
G-418 Calibration
- In 96-well assay plate in well 1 add 90 ul of yeast BY4735 strain into the well plate with 10 ul of water for the control.
- In the same well plate in well 2 add 90 ul of yeast BY4735 into the well plate with 10 ul of Geneticin (also known as G-418) with a concentration of 100 ul/ml
- In the same well plate in well 3 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 50 ul/ml
- In the same well plate in well 4 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 25 ul/ml
- In the same well plate in well 5 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 12.5 ul/ml
- In the same well plate in well 6 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 6.3 ul/ml
- In the same well plate in well 7 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 3.1 ul/ml
- In the same well plate in well 8 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 1.56 ul/ml
- In the same well plate in well 9 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 0.78 ul/ml
- In the same well plate in well 10 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 0.39 ul/ml
- In the same well plate in well 11 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 0.2 ul/ml
- In the same well plate in well 12 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 0.1 ul/ml
- Next, was the UW-Stout/Growth Curve process
Calibration Data
From the graphs, we determined that 100 ul/ml was the best option to use for our knock-out experiment because it slowed down the growth of BY4735 without killing the yeast.
Knock-out Experiment
- In 96-well assay plate in well 1 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- In same well assay plate in well 2 add 90 ul of yeast YEL035C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- In same well assay plate in well 3 add 90 ul of yeast YCL002C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- In same well assay plate in well 4 add 90 ul of yeast YFL064C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- In same well assay plate in well 5 add 90 ul of yeast YGL235W into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- In same well assay plate in well 6 add 90 ul of yeast YCR100C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- In same well assay plate in well 7 add 90 ul of yeast YJL043W into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- In same well assay plate in well 8 add 90 ul of yeast YPL068C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- In same well assay plate in well 9 add 90 ul of yeast YMR090W into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- Next was the UW-Stout/Growth Curve process