Difference between revisions of "UW-Stout/Caffeine SP21"

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== Introduction ==
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The variable I will be testing is caffeine and how it affects the growth of yeast cells. I will be varying the concentration of caffeine exposed to the yeast through dilution with water. The control will be a well that has no caffeine present in it, just 50 ul of water mixed with my yeast. This process will help me figure out which concentration of caffeine works best for my next experiment with the KO genes that stresses the yeast enough but does not kill them all off.
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== Safety ==
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Basic safety precautions while performing this experiment, wear gloves and lab coat and safety goggles.
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== Materials ==
 
== Materials ==
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== Data ==
 
== Data ==
We settled on the concentration of 1.75 mM. In regards to the graph this is the burgundy colored line, it had the best growth curve represented in our data. Other concentrations the yeast did not survive at all and showed no growth curve. The concentration of 0.875 also did grow but not as successfully as the concentration of 1.75 mM.
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We settled on the concentration of 1.75 mM. In regards to the graph this is the burgundy colored line, it had the best growth curve represented in our data. Other concentrations the yeast did not survive at all and showed no growth curve. The concentration of 0.875 mM also did grow but not as successfully as the concentration of 1.75 mM.
 
[[Image:calibrationconcentrationyeastexperimentgraph.jpg|center|thumb|500px|Calibration Curve]]
 
[[Image:calibrationconcentrationyeastexperimentgraph.jpg|center|thumb|500px|Calibration Curve]]
  
 
== Protocol for KO Genes ==
 
== Protocol for KO Genes ==
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== Introduction ==
 +
I will take the concentration at which I did see there to be an effect on the growth of the yeast yet did not kill all of my yeast cells off. I will then use this concentration to test with the knockout gene yeast to see the growth curve. Will test to see if there is a difference between my knockout gene yeast and the wild type strain.
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 +
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== Safety ==
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Basic safety precautions while performing this experiment, wear gloves and lab coat and safety goggles.
 +
 +
 
#Get master solution out of the fridge.   
 
#Get master solution out of the fridge.   
 
#Swirl test tube until no chunks are left in the solution.  
 
#Swirl test tube until no chunks are left in the solution.  

Latest revision as of 10:42, 27 April 2021


Introduction

The variable I will be testing is caffeine and how it affects the growth of yeast cells. I will be varying the concentration of caffeine exposed to the yeast through dilution with water. The control will be a well that has no caffeine present in it, just 50 ul of water mixed with my yeast. This process will help me figure out which concentration of caffeine works best for my next experiment with the KO genes that stresses the yeast enough but does not kill them all off.


Safety

Basic safety precautions while performing this experiment, wear gloves and lab coat and safety goggles.


Materials

  • Caffeine 80 mM solution in water. (15.52 x 25 ml water= .388 g of caffeine)
  • Sterile H2O
  • Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
  • Corning COSTAR 96-well clear flat-bottom assay plate
  • 250 mL beaker for caffeine master solution.
  • P-20,200 micro pipette/ tips
  • Scale
  • Tube to store caffeine
  • Powdered caffeine for solution


Equipment

  • Molecular Devices SpectraMax Plus 384 Microplate Reader


Protocol

  1. Weigh out .388 grams of caffeine.
  2. Add to 25 ml of water in test tube and swirl until it’s mixed with no chunks.
  3. Set up 8 test tubes and label them 1 through 8 matching with the order of wells.
  4. Using the p20 pipette, pipette the varying amounts of caffeine solution provided in table.
  5. Then using a p200 pipette, pipette the varying amount of sterile water into the provided tubes.
  6. When under the hood add 50 ul of yeast to each test tube except for the tube labeled 0 which is the control.
  7. Use the table below to put the correct concentrations in each tube

Well 0 (control)

  • 0 ul caffeine
  • 50 ul yeast
  • 50 ul water

Well 1

  • 50 ul caffeine
  • 50 ul yeast
  • 0 ul water

Well 2

  • 25 ul caffeine
  • 50 ul yeast
  • 25 ul water

Well 3

  • 12.5 caffeine
  • 50 ul yeast
  • 37.5 ul water

Well 4

  • 6.2 ul caffeine
  • 50 ul yeast
  • 43.8 ul water

Well 5

  • 3.1 ul caffeine
  • 50 ul yeast
  • 46.9 ul water

Well 6

  • 2.2 ul caffeine
  • 50 ul yeast
  • 47.8 ul water

Well 7

  • 1.1 ul caffeine
  • 50 ul yeast
  • 48.9 ul water


  1. Transfer the solution created in each tube into the wells, writing down which row your wells are.
  2. Store extra master caffeine solution in a labeled tube in the fridge.
  3. Set up the plate reader as follows:
  • Temperature: 30°C
  • Mode: Kinetic
  • Wavelength: 600 nm
  • Interval: 10 minutes
  • Total run time: 12 hours
  • Shake before read: 30 seconds
  • Transfer the assay plate to the reader and read for 24 hours.

This process will be replicated 3 times.


Data

We settled on the concentration of 1.75 mM. In regards to the graph this is the burgundy colored line, it had the best growth curve represented in our data. Other concentrations the yeast did not survive at all and showed no growth curve. The concentration of 0.875 mM also did grow but not as successfully as the concentration of 1.75 mM.

Calibration Curve

Protocol for KO Genes

Introduction

I will take the concentration at which I did see there to be an effect on the growth of the yeast yet did not kill all of my yeast cells off. I will then use this concentration to test with the knockout gene yeast to see the growth curve. Will test to see if there is a difference between my knockout gene yeast and the wild type strain.


Safety

Basic safety precautions while performing this experiment, wear gloves and lab coat and safety goggles.


  1. Get master solution out of the fridge.
  2. Swirl test tube until no chunks are left in the solution.
  3. Set up 8 test tubes and label them 0 through 5 matching with the order of wells.
  4. Using the table before pipette the correct concentrations of each solution into each tube.

Well 0

  • 0 ul caffeine
  • 50 ul yeast
  • 50 ul water

Well 1 YOR387C

  • 2.2 ul caffeine
  • 50 ul yeast
  • 47.8 ul water

Well 2 YKL12JW

  • 2.2 ul caffeine
  • 50 ul yeast
  • 47.8 ul water

Well 3 YBR225W

  • 2.2 ul caffeine
  • 50 ul yeast
  • 47.8 ul water

Well 4 YNL018C

  • 2.2 ul caffeine
  • 50 ul yeast
  • 47.8 ul water

Well 5 YLR426W

  • 2.2 ul caffeine
  • 50 ul yeast
  • 47.8 ul water

Well 6 YGL138C

  • 2.2 ul caffeine
  • 50 ul yeast
  • 47.8 ul water
  1. Transfer the solution created in each tube into the wells, writing down which row your wells are.
  2. Set up the plate reader as follows:
  • Temperature: 30°C
  • Mode: Kinetic
  • Wavelength: 600 nm
  • Interval: 10 minutes
  • Total run time: 12 hours
  • Shake before read: 30 seconds
  • Transfer the assay plate to the reader and read for 24 hours.

This process will be replicated 3 times.