Difference between revisions of "Endy:F2620/Latency/Protocols"
Revision as of 09:13, 5 September 2006
- One culture, inoculated from a single colony, was grown for 15hrs in M9salts(Bio101Inc.) with 0.005%(w/v) Casamino acids, 0.1%(v/v)glycerol, 2nMMgSO4, 0.1mM CaCl2 and kanamycin(20μg/ml) at 370C with shaking at 70rpm. [Standard overnight culture]
- Culture was diluted into fresh medium and allowed to grow for an additional 5 hours under the same conditions. 1:1000 dilution i.e (5uL into 5mL of M9+KAN)
- 200μ l of the culture was transferred into flat-bottom 96 well plates (Greiner). Wells were pre-filled with AHL of final concetration 10μM 2uL of stock solution of 1mM AHL.
- 3x Well with AHL at t=0
- 3x Well with AHL at t=0 and add RifAmo at t=20min (RifAmp stock solution in the -20 fridge)
- 3x Well with AHL at t=0 and RifAmp added at t=0
- 3x Well with cell and no AHL
[we did ethanol control before but I don't think we need to repeat it anymore]
- Run the Latency protocol first and then after 20min RifAmp protocol on the plate reader (you need to check which wells will it add RifAmp to and then position them appropriatelly)