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

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*Eight different cultures, each inoculated from a single colony were 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.  
#Prepare 6 cultures for testing using the [[Endy:F2620/Standard chassis preparation|standard chassis preparation]] protocol.
*Cultures were diluted into fresh medium and allowed to grow for an additional 5 hours under the same conditions.
#Transfer the appropriate number of 200μl aliquots of each of the cultures into a flat-bottom 96 well plate (Cellstar Uclear bottom, Greiner).
*200μ l of the culture was transferred into flat-bottom 96 well plates (Greiner). Wells were pre-filled with the appropriate inducer molecule.  
#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 used to grow the cultures in a Wallac Victor3 multi-well fluorimeter at 370 C 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 a background measurement. Time between measurements was 3 minutes.
#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.
*Each of the 8 cultures were induced with 8 concentrations of AHL (0nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, 1μ M, 10μ M).

Latest revision as of 12:38, 2 June 2007

  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.
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