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I13207 AiiA Test Construct Protocol

  • Culture bacteria (DH5a) in LB medium, incubate overnight
  • Transfer plasmids with desired part via electroporation
  • Incubate overnight
  • Transfer colonies into liquid medium (amp resistant, 2ml)
  • Wait until optical density is 0.1
  • Make 20mL of 1% arabinose LB solution
  • Then make the following solutions:
Arabinose Solutions
Concentration How to set up solutions
1.00% 10mL 1.00% arabinose solution as made up above
0.50% 5 mL 1.00% and 5 mL Pure LB
0.10% 1 mL 1.00% and 9 mL Pure LB
0.05% 1 mL 0.05% and 9 mL Pure LB
0.00% 10 mL Pure LB
  • Transfer arabinose solutions into 5 different labeled tubes
  • One well is filled with pure LB media in all experiments as a control
  • Transfer 2 mL of each arabinose solution into separate clear tubes
  • Add 200ul of DH5a culture into the tubes
  • Assay 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)
  • Shake (1 mm, linear, normal speed)
  • Time between measurements should be 20 minutes
  • Time period for measurements should be ~5 h
  • Perform 2 tests: one with 0.5ml of 0.5M ZnCl2 in the LB medium and one without