McClean:Phase Plane Mapping

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Purpose

Expose a strain of yeast to all combinations of two ranges of stimulants, in this case alpha-factor and sorbitol, and measure their response with fluorescent markers and flow cytometry.


Protocol

  1. Fill falcon tubes with 800 microL PBS + .1% tween, put on ice
  2. Grow up 25ml of yMM736 to mid-log phase in a klett flask.
  3. Plan the conditions to test:
    • control
    • sorbitol: 0M, .25M, .5M, .75M, 1M
    • alpha-factor: 0ng, 10ng, 100ng, and 1μg (per mL)
  4. Prepare a test tube for each condition. To control for cell density prepare each tube with an equal volume of LFM + sorbitol
    • Sorbitol
      • 0M: 1 ml LFM
      • 0.25M: .75 ml LFM + .25 ml LFM + 2M sorbitol
      • 0.5M: .5 ml LFM + .5 ml LFM + 2M sorbitol
      • 0.75M: .25 ml LFM + .75 ml LFM + 2M sorbitol
      • 1M: 1 ml LFM + 2M sorbitol
    • alpha-factor: add 2 μL of alpha-factor that is at 1000x concentration, and use straight DMSo for the 0 ng/mL control.
  5. At t=0 minutes pipette 1 mL of culture into each condition tube
  6. At t=10 minutes add 2 ml of 50 μg/mL cycloheximide to a final concentration of 100 microg/ml
  7. Take 250 microL samples at 3 hours after induction, add these to the ice cold PBS + tween.
  8. Sonicate samples briefly
  9. FACS sort samples
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