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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 1:500 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.
- Aliquot 200 μl of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:
- temperature: 37°C
- sampling time: 5 minutes
- 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
- fluorescence gain: 50 - 80
- O.D. filter: 600 nm
- RFP filters: 535 nm (excitation) / 620 nm (emission)
- duration time: 10 - 15 hours
pLux 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 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours.
- 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.
- Aliquot 200 μl of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:
- temperature: 37°C
- sampling time: 5 minutes
- 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
- fluorescence gain: 50 - 80
- O.D. filter: 600 nm
- RFP filters: 535 nm (excitation) / 620 nm (emission)
- duration time: 10 - 15 hours
Enzyme activity assay
AiiA activity
