McClean:Fluorescent Protein Start

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.