Endy:F2620/Transfer Curve/Protocols

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  1. Prepare 6 cultures for testing using the standard chassis preparation protocol.
  2. Transfer the appropriate number of 200μl aliquots of each of the cultures into a flat-bottom 96 well plate (Cellstar Uclear bottom, Greiner).
  3. Each of the 6 cultures were induced with 8 concentrations of AHL (0nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, 1μ M, 10μ M). Each of these measurements were performed on three replicates for each culture.
  4. These plates were incubated in a Wallac Victor3 multi-well fluorimeter at 37C and assayed with an automatically repeating protocol of absorbance measurements (600nm filter, 0.1 second absorbance through approximately 0.5cm of fluid), fluorescence readings (488nm excitation filter, 525nm emission filter, 0.5 second, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed). One well was filled with M9 media in all experiments as an absorbance background. Time between measurements was 3 minutes.