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

• IGEM:IMPERIAL/2006/LabCalendar/2006-8-23 Events
• Day in the lab
• Project Progress

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