IGEM:IMPERIAL/2006/LabCalendar/2006-8-23

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Contents

S01656 Testing

  • Measure OD of cultures
    • +IPTG = 1.735
    • -IPTG = 0.298 (lack of oxygen in culture, since cultured 50 mL in 50 mL Falcon tube)
  • Dilute down to OD 0.1 in 5 mL LB Kanamycin
  • Start shake at 9:25
  • Measure OD of cultures again
    • +IPTG = 0.389
    • -IPTG = 0.390
  • Dilute down to OD 0.1 in 5 mL LB (no antibiotic)
  • Start shake at 11:40
  • Measure OD of T9002
    • OD = 0.374
  • Spun down S01656 cultures, put into T9002, placed into 96 well plate
  • Start shake at 14:07

J37015 + J37015 RS Testing

  • Inoculated fresh day cultures at 9.40 am


T9002 & J37016 Testing

  • Measurement of OD in the morning:
    • T9002: 0.549
    • J37016: 0.676
  • Dilution to OD 0.1 & put in shaker at 9:45 am
  • Measurement of OD after 2h in shaker:
    • T9002: 0.359
    • J37015: 0.433
  • Dilution to OD 0.1 again - then transferring solutions with appropriate concentration of AHL respectively into 96 well plate
  • Put in shaker at 12:30 pm (set shaker to 100rev/min otherwise platees might get contaminated)
  • After 4:45 h took plates out of shaker and read fluorescence with Wallac Victor III

LoxP PCR

  • Inconclusive results - further tests tomorrow
  • The problem that we have is that the amplified bands are of a very similar size to that of the oligonucleotides, and therefore distinguishing them is proving difficult.
  • We are altering the compositions of the gels as a consequence.

Storing Bacteria

  • Following the MIT protocol S01656, T9002, J37016, J37015 & J37015RS have been stored in the -80C freezer. (They were stored with an initial OD of 0.5)

Cultures

  • Cultures set up for testing tomorrow:
    • 2μL S01656 in 2ml LB KAN (-IPTG)
    • 2μL S01656 in 2ml LB KAN (+ 20μL IPTG)
    • 2μL T9002 in 2ml LB AMP
    • 2μL J37016 in 2ml LB AMP
    • 2μL J37020 in 2ml LB AMP
    • 2μL J37015 in 2ml LB AMP
    • 2μL J37015RS in 2ml LB AMP
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