Team:UNIPV-Pavia/Measurements

pTet protocol

1. Streak long term storage glycerol stocks on a LB agar plate + Cm12.5 (don't forget positive and negative controls). Let them grow over night.
2. Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night.
3. Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours.
4. Induce cultures in falcon tube with anhydrotetracycline (aTc) (final concentrations: 0 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 8 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml). Grow cultures for three hours.
5. Aliquote cultures in Tecan Infinite F200 microplate reader, filling each well with 200 μl of cultures. Set the automatic procedure:
   • temperature: 37°C
   • sampling time: 5 minutes
   • fluorescence gain: 50
   • O.D. filter: 600 nm
   • RFP filters: 535 nm (excitation) / 620 nm (emission)
   • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
   • duration time: 10 - 15 hours

pLux protocol

1. Streak long term storage glycerol stocks on a LB agar plate + Cm12.5 (don't forget positive and negative controls). Let them grow over night.
2. Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night.
3. Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours.
4. Induce cultures in falcon tube with 3OC₆-HSL (final concentrations: 0 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, 100 nM). Grow cultures for three hours.
5. Aliquote cultures in Tecan Infinite F200 microplate reader, filling each well with 200 μl of cultures. Set the automatic procedure:
   • temperature: 37°C
   • sampling time: 5 minutes
   • fluorescence gain: 50
   • O.D. filter: 600 nm
   • RFP filters: 535 nm (excitation) / 620 nm (emission)
   • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
   • duration time: 10 - 15 hours