Difference between revisions of "UW-Stout/G-418"
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+ | ==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==== | ||
+ | BY4735 (wild type), | ||
+ | YEL035C, | ||
+ | YCL002C, | ||
+ | YFL064C, | ||
+ | YGL235W, | ||
+ | YCR100C, | ||
+ | YJL043W, | ||
+ | YPL068C, and | ||
+ | YMR090W | ||
+ | |||
+ | ====Equipment==== | ||
+ | * Micropipettes | ||
+ | |||
==G-418 Calibration== | ==G-418 Calibration== | ||
− | # In 96-well assay plate in well 1 add 90 ul of yeast BY4735 into the well plate with 10 ul of water for the control. | + | # In 96-well assay plate in well 1 add 90 ul of yeast [https://www.atcc.org/en/Products/All/200897.aspx 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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/ml |
− | + | # Next, was the [[UW-Stout/Growth Curve]] process | |
− | ==Calibration Data== | + | ===Calibration Data=== |
− | From the graphs, we determined that 100 | + | From the graphs, we determined that 100 mg/ml was the best option to use for our knock-out experiment because it slowed down the growth of BY4735 without killing the yeast. |
− | [[File:Calibration graphs_5889_image014.png | + | [[File:Calibration graphs_5889_image014.png|This graph shows the results of testing the absorbance of yeast with different amounts of G-418.]] |
==Knock-out Experiment== | ==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 | + | # 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 mg/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 | + | # 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 mg/ml |
− | # In same well assay plate in well 3 add 90 ul of yeast | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/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 | + | # 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 mg/ml |
+ | # Next was the [[UW-Stout/Growth Curve]] process |
Latest revision as of 07:13, 10 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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/ml
- Next, was the UW-Stout/Growth Curve process
Calibration Data
From the graphs, we determined that 100 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/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 mg/ml
- Next was the UW-Stout/Growth Curve process