McClean:Induction Timecourse

Purpose

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.

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 (aim for klett 50-60). Have one klett flask for each condition to test.
3. 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.
4. At t = -2 minutes take 1 ml samples and add to appropriate eppendorf, pipetting up and down to mix.
5. 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
6. 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.
7. Exactly 3 hours after each timepoint take 250 μl of the eppendorf sample and add it to a tube of PBS + tween.
8. Sonicate samples briefly
9. Measure samples with flow cytometry