UW Stout/Ethanol FA19

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Yeast torture 1

Protocol:

Materials:

  1. neat ethanol
  2. yeast (WT:BY4735)
  3. sterile water
  4. 200 ul
  5. 96 well plate

Procedure:

  1. (add 50ul mixture to 50ul yeast in 96 well plate.)x2. Used 12 wells in total
  2. fill 6 200ul tubes, each with water and ethanol concentration x2. Resulting in 6 200ul tubes with 100ul of mixture in each.
1 2 3 4 5 6
ul ethanol 0 3.75 7 10.25 13.5 16.75
ul water 50 46.25 43 39.75 37.5 33.25
ul yeast 50 50 50 50 50 50

BY4735.et.graph.png

This graph shows the ethanol tolerance in the Wild Type Yeast Cells (BY4735). This graph helped us determine the amount of yeast to add to our knockout strains. We determined this by examining the graph and found a percentage of ethanol that shows a change in growth and doesn't kill the cells. For this we chose the 7% ethanol to use on our yeast knockout strands.

Knockout Strand Yeast Torture

Protocol:

Materials:

  1. Yeast Knockout Strains:
    1. YAL061W
    2. YBL086C
    3. YBL9081W
    4. YBR033W
    5. YBR138C
    6. YBR220C
    7. YHR048W
    8. YGL140C
    9. YJR061W
    10. YKR005C
    11. WT:BY4735
  2. neat ethanol
  3. sterile water
  4. micropipeter(s)
  5. 96 well plate

Procedure:

  1. add 50 ul of Knockout Strain to individual well (96 well plate).
  2. add 50 ul of water+ethanol mixture to individual well (Follow table below).
Well 1 2 3 4 5 6 7 8 9 10 11 12
ul yeast 50 50 50 50 50 50 50 50 50 50 50 50
ul ethanol 7 7 7 7 7 7 7 7 7 7 7 7
ul water 43 43 43 43 43 43 43 43 43 43 43 43