IGEM:PennState/2006/Motility-assays
From OpenWetWare
Jump to navigationJump to search
Swarm Plate Assays
Swarm Plate Assays
Time: 2 hr first day; 9 hr second day
Reference: Garza, A.G., et al. J. Mol. Biol. (1996) 258, 270-285
Media/Reagents/Materials
- LB
- Typtone broth plates
- Cells (from plate)
Day 1: Make Tryptone Swarm Plates
1. For each plate to be made, add the following to a 125mL flask:
- 0.25g Bacto tryptone
- 0.20g NaCl
- 0.15g sodium citrate
- 0.05g Eiken agar (for a 0.20% agar swarm plate)
2. Add 25mL purified water to the flask and swirl to dissolve
3. Autoclave 25 minutes with slow exhaust
4. Upon cooling, add appropriate antibiotics and inducers
- IPTG saturation concentration => 1mM
- HSL saturation concentration => 100µM
5. Allow plates to dry overnight
Day 1: Inoculate O'N Culture
1. Inoculate 3mL overnight cultures of all samples to be tested
2. Shake at 200 RPM in 30°C incubator
Day 2: Assay
1. Dilute overnight culture 1:100 in LB (30uL of culture in 3mL LB)
2. Grow in a 30°C incubator at 200 RPM to an A590nm O.D. between 0.3-0.4
3. Add about 3uL of each culture to plates
4. Grow in a 30°C incubator 8 hours
5. Take picture of plate with digital camera
