Endy:F2620/Transfer Curve/Protocols: Difference between revisions

Latest revision as of 12:38, 2 June 2007

Prepare 6 cultures for testing using the standard chassis preparation protocol.
Transfer the appropriate number of 200μl aliquots of each of the cultures into a flat-bottom 96 well plate (Cellstar Uclear bottom, Greiner).
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.
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.

@@ Line 1: / Line 1: @@
-#Prepare 6 cultures for testing using the [[Endy:F2620/Standard chassis preparation|standard chassis prepartion]] protocol.
+#Prepare 6 cultures for testing using the [[Endy:F2620/Standard chassis preparation|standard chassis preparation]] protocol.
-#The standard protocol was modified by pre-adding the appropriate concentration of [[Endy:F2620/AHL|AHL]] to the wells.  Each of the 3 replicates of the 6 cultures were induced with 8 concentrations of AHL (0nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, 1μ M, 10μ M).
+#Transfer the appropriate number of 200μl aliquots of each of the cultures into a flat-bottom 96 well plate (Cellstar Uclear bottom, Greiner).
+#Each of the 6 cultures were induced with 8 concentrations of [[Endy:F2620/AHL|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.
 #These plates were incubated in a [[Endy:Victor3 plate reader|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.