4 - Lac Operon

4 - Lac Operon kdorfman Fri, 08/07/2020 - 15:45

Qualitative lac operon experiment

Grow GFP E. coli in different sugar media, then photograph in a 12 well plate on a blue light box through the orange filter.

  • Streak GFP E. coli on an LB-glucose-kan plate, grow overnight

  • Pick a colony, grow in overnight in liquid LB-glucose-kan (to prevent it turning green)

  • Spin down the ON culture (take a picture of the pellet for other use!)

  • Rinse several times (to get rid of the glucose)

  • Fill 12 well plate as indicated below. 2 mL per well:

additive none IPTG 0.01mM lac 0.25M gal 0.25M
glu 0.25M
mal 0.25M
  • Photograph
    • in white light
    • in blue light
  • Read in plate reader for blanks (OD and FL)

    • 12-well plate is too tall for reader!!!
    • Sample 100 uL of each to read blanks (OD & FL in a 96 well plate)
  • Inoculate each well with a small amount of culture.

  • Incubate on the shaker.

  • Photograph

    • in white light
    • in blue light
  • Read in plate reader (OD and FL)
    • Take 100 uL (?) samples to read in 96 well plate


Need enough for the qualitative experiment ~5 mL each

Need enough for the quantitative experiment: 96 well plate @ 250 uL/well = ~25 mL total

Make 25 mL of each high concentration stock; mix the combinations from that

Add 50 µg/mL Kanamycin as indicated here.

Lac Operon PowerPoint from 2020 remote class