Experimenting with AHL

From FreeBio

1. Inoculate overnight cultures in media with appropriate antibiotics; don't forget positive and negative controls.

2. Backdilute cultures to OD600 = 0.1 with LB + antibiotic; make enough cell solution to aliquot 2 ml for each concentration of AHL being tested. I find it helpful to have 2 ml per concentration plus 2 extra; when experimenting with 3 concentrations of AHL, for example, I would make a total of 8 ml of 0.1 OD600 solution. -- Connie

4. If there are any strains with the Kan plasmid, save 2 ml after backdiluting. Add 2 mM IPTG and incubate in 37 C shaker for 2 hours before rebackdiluting and proceeding with protocol.

3. Aliquot 2 ml of solution into tubes.

4. Add 20 ul of AHL solution at appropriate concentrations.

5. Incubate in 37 C shaker for 40 minutes.

6. Put 500 ul of each culture into an eppendorf; spin down and aspirate.

7. Resuspend cell pellet in 15 ul of LB.

8. Place 1 ul of solution on slide.

9. Look at cells under microscope.

• Note: Current AHL stock found in the refridgerator is 1000X = 492 uM. Dilute 1:10 to create 100X stock, then make serial dilutions. The concentrations suggested by the Weiss paper are 0 nM, 15 nM, 50 nM, 150 nM, 500 nM. Perhaps try 5000 nM in the future?