"
Contents |
pTet protocol
- 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.
- Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night.
- Dilute cultures 1:100 in 1 ml of M9 + Cm12.5 and let them grow for three hours.
- 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.
- 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
