Endy:F2620/Latency/Protocols: Difference between revisions

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*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]''
#Prepare 1 culture for testing using the [[Endy:F2620/Standard chassis preparation|standard chassis prepartion]] protocol.
*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)''
#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 AHL of final concetration 10μM ''2uL of stock solution of 1mM AHL''.  
#Induce three replicates of the culture with an input of 100nm [[Endy:F2620/AHL|AHL]], sufficient for full induction of the device.  Leave three replicates of the culture uninduced.  Three replicates of a media control are used to measure absorbance background.
**3x Well with AHL at t=0
#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 ~54s.
**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 [we added 5uL to make sure]
**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)

Latest revision as of 11:32, 14 September 2006

  1. Prepare 1 culture for testing using the standard chassis prepartion 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. Induce three replicates of the culture with an input of 100nm AHL, sufficient for full induction of the device. Leave three replicates of the culture uninduced. Three replicates of a media control are used to measure absorbance background.
  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 ~54s.
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