IGEM:IMPERIAL/2007/Projects/Hrp System/Systems/Hrp Device 1/TestingValidation/Protocol

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Preparing the Over night Cultures

  • Inoculate a culture from 10μl of stored culture in 5ml M9 minimal media containing 40 μg/mL Ampicillin.
  • Incubate at 37°C for overnight in a shaker.
    • Allows selected growth of E.coli with vector

Day 1

  • Incubate M9 Amp Media 37o
  • Remove the culture and measure O.D.600.
  • We want to inoculate 10ml of M9 minimal media to give an O.D.600 of 0.1, to do this we calculate the ratio of volumes of:
    • o/n culture in M9 media
    • Pre-warmed M9 media
  • Use simple calculate of: volume o/n culture = (0.1/O.D.600)*10ml
  • This will calculate volume of o/n culture needed, this is then made up to 10ml with pre-warmed M9 media
  • Then take the culture and inoculate pre warmed fresh culture of M9 medium to an O.D.600 of 0.1.
  • Return the 10ml culture to the incubator and incubate at 37oC for 2 hours in a shaker.
    • The purpose of inoculating a new media is because o/n they go into the stationary phase, but by inoculating a new media are returned to exponential phase

2 hours Incubation

  • After this, remove culture measure and record the OD600.
  • Dilute again for an O.D.600 of 0.1 in a new culture of 15ml of a prewarmed M9 Ampicilin. (same principle as above)
  • Spin down the cultures, retrieve cells and dispose of the supernatant
  • To the cells add 15ml of prewarmed M9 Amp.
    • This is to supply fresh media, ensures the cultures are more homogenous and that the cells are in exponential phase

Plate Reader

  • Pipette samples into a 96 well plate
  • Add 200μL of growth medium to a well to act as a control
  • Add 8 x 200μL of the cultures to the well.
  • These 8 wells should then have 2ul of the inducer concentrations to each of the well.
  • In addition want two controls:
  1. 200μl of growth medium.
  2. Back ground fluorescence.