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.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]  
+
* [https://www.invivogen.com/g418 Geneticin 100 mg/ml]  
# Yeast
+
* Glycerol stocks of [[UW-Stout/Knockout_Protocol|knockout strains]]
# Water
+
* Water
 +
====Strains of Yeast====
 +
BY4735 (wild type),
 +
YEL035C,
 +
YCL002C,
 +
YFL064C,
 +
YGL235W,
 +
YCR100C,
 +
YJL043W,
 +
YPL068C, and
 +
YMR090W
  
==Equipment==
+
====Equipment====
# Micropipettes
+
* Micropipettes
# 96-well assay plate
 
  
 
==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 ul/ml
+
# 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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.
  
==Calibration Data==
+
[[File:Calibration graphs_5889_image014.png|This graph shows the results of testing the absorbance of yeast with different amounts of G-418.]]
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.
 
 
 
[[File:Calibration graphs_5889_image014.png|thumb|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 ul/ml
+
# 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 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 mg/ml
# In same well assay plate in well 3 add 90 ul of yeast YCL200C 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 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 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 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 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 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 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 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 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 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 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 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 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 mg/ml
 +
# Next was the [[UW-Stout/Growth Curve]] process

Latest revision as of 07:13, 10 May 2019

Materials

Strains of Yeast

BY4735 (wild type), YEL035C, YCL002C, YFL064C, YGL235W, YCR100C, YJL043W, YPL068C, and YMR090W

Equipment

  • Micropipettes

G-418 Calibration

  1. 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.
  2. 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
  3. 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
  4. 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
  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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. 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
  12. 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
  13. 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.

This graph shows the results of testing the absorbance of yeast with different amounts of G-418.

Knock-out Experiment

  1. 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
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. Next was the UW-Stout/Growth Curve process