SEED/2010/Day 8

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Contents

Morning

  • Discuss beta-gal assay and AHL-inducible lacZ
  • Take 20ul of the culture into 80ul of the permeabilization solution (directly in cuvettes)
  • Leave at 30C over lunch
  • Measure the OD600 of the original cultures while waiting

Afternoon

  • Add 600ul substrate solution to permeabilized cells
  • Every 20 minutes, make another measurement and record time

Homework

  • Analyze data
  • Plot results
  • Compare results with existing F2620 data

Instructor Pre-prep

  • grow up 1ml cultures expressing F2620.lacZa (BBa_T9003) with IPTG and different amounts of AHL
    • 0, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10, 1E-11
  • grow up 1ml control cultures of cells with and w/o lacZa expression plasmid
  • Make solutions as specified in Beta-Galactosidase_Assay_(A_better_Miller)
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