McClean:Fluorescent Protein Start
Find the earliest detectable fluorescent protein after induction.
- Fill falcon tubes with 800 microL PBS + .1% tween, put on ice
- Grow up 25ml of yMM736 to mid-log phase in a klett flask.
- Take an initial 250 microL sample of the culture
- Alloquot 2ml of culture into 10 different test tubes and put on rollers, labeled as follows:
- 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
- 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
- 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)
- wait until 60 minutes post addition of cycloheximide
- 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.
- FACS sort samples
- Adjust the time points and the time to wait for folding based on what you know about your specific promoter and fluorescent protein.