McClean:Fluorescent Protein Start

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Purpose

Find the earliest detectable fluorescent protein after induction.


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. Take an initial 250 microL sample of the culture
  4. Alloquot 2ml of culture into 10 different test tubes and put on rollers, labeled as follows:
    • original
    • no alpha add cyclo at 0 min
    • no alpha no cyclo
    • alpha only
    • -10 min
    • -5 min
    • 0 min
    • 5 min
    • 10 min
    • 15 min
    • 20 min
    • 30 min
  5. Add 4 microL of 50mg/ml cyclohexamide to each tube for a final conc of 100microg/ml cyclo at the give time before or after the addition of alpha factor
  6. Add alpha factor to each tube, 2 microL of 100 microg/ml alpha will give 100 ng/ml alpha. Alpha factor added at 0 minutes. (alternativel 0.2 microL of 1mg/ml alpha)
  7. wait until 60 minutes post addition of cycloheximide
  8. take second time points from each tube (250 microL samples into appropriate falcon tubes)
    • For the time points above the second time points will be at 50, 55, 60, 65, 70, 75, 80, and 90 minutes.
  9. FACS sort samples


Notes

  • Adjust the time points and the time to wait for folding based on what you know about your specific promoter and fluorescent protein.