UW-Stout/Caffeine SP21

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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. At a couple other concentrations the yeast did grow but not as successfully as the concentration of 1.75 mM. calibrationconcentrationyeastexperimentgraph.jpg

Protocol for KO Genes

  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.