This is a protocol to measure transcriptional response to a stimulant over time. Cycloheximide is used to halt translation and allow all already translated fluorescent proteins to fully fold before measuring the cells with flow cytometry.
- 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 (aim for klett 50-60). Have one klett flask for each condition to test.
- Prepare eppendorfs containing 0.1 ml of LFM + 2.2 μl of 50 mg/ml cyclohexamide. It's easier to make a master mix and alloquot it into the eppendorfs.
- At t = -2 minutes take 1 ml samples and add to appropriate eppendorf, pipetting up and down to mix.
- At t = 0 minutes induce the flask cultures with the appropriate stimulant. For the 8/4 experiment, add:
- control: 0M sorb, 0 ng/ml alpha: 8 ml of LFM + 32 μl of 0 μg/ml alpha-factor
- alpha only: 0M sorb, 10 ng/ml alpha: 8ml of LFM + 32 μl of 10 μg/ml alpha
- sorb only: 0.5M sorb, 0 ng/ml alpha: 8ml of LFM + 2M sorb + 32 μl of 0 μg/ml alpha-factor
- alpha + sorb: 0.5M sorb, 10 ng/ml alpha-factor: 8ml of LFM + 2M sorb + 32 μl of 10 μg/ml alpha-factor
- At each timepoint ( t = 10, 20, 30, 40, 50, 60, 70, 80, 90) take a 1 ml sample of the culture and add it to the appropriate eppendorf. Pipet up and down to mix.
- Exactly 3 hours after each timepoint take 250 μl of the eppendorf sample and add it to a tube of PBS + tween.
- Sonicate samples briefly
- Measure samples with flow cytometry