TITLE: FLUORESCENCE LOSS ASSAY
AUTHORS: Alexander Niederau, Despoina Trasanidou

[STEPS]
?. [Day 1 (=Transformation -> ONC in 96-well plate)]
50ul of chemically competent E. coli DH10B_gfp cells were transformed (heat-shock: 42°C, 30sec) with 3ng plasmid (Cplasmid*2ul=3ng/ul*x -> x=...ul=2ul undiluted plasmid + rest ul MQ) and recovered in 450 ul LB for 1h at 37°C. (PC = E. coli DH10B_gfp + pACYC184) (NC = E. coli DH10B + pACYC184)
?. [Day 1 (=Transformation -> ONC in 96-well plate)]
2ul of recovered cells were inoculated in 198ul M9TG+Cam15. [1 Masterblock] (B = Blank, just medium)
?. [Day 1 (=Transformation -> ONC in 96-well plate)]
Incubate at 37°C under shaking (900rpm) for 21-22 hours.
?. [Day 2 (=Dilution 1/100 NO INDUCERS -> Dilution 1/100 WITH INDUCERS -> ONC in triplicates 96-well plates)]
2ul of each preculture were inoculated in 198ul M9TG+Cam15. [1 Masterblock]
?. [Day 2 (=Dilution 1/100 NO INDUCERS -> Dilution 1/100 WITH INDUCERS -> ONC in triplicates 96-well plates)]
Shake the plate on the thermoblock (900rpm)
?. [Day 2 (=Dilution 1/100 NO INDUCERS -> Dilution 1/100 WITH INDUCERS -> ONC in triplicates 96-well plates)]
2ul of each dilution were inoculated in 198ul M9TG+Cam15+inducer. [4 Masterblocks=triplicates + control plate]  Plate 1,2,3 (triplicates)
?. [Day 2 (=Dilution 1/100 NO INDUCERS -> Dilution 1/100 WITH INDUCERS -> ONC in triplicates 96-well plates)]
Incubate at 37°C under shaking (900rpm) for 21-22 hours.
?. [Day 3 (=SPECTROPHOTOMETRY: 1/5 DILUTION -> 100ul)]
Mix at thermoblock the preculture -> 40ul of each sample were inoculated in 160ul of 1xPBS [4 normal 96-well plates]
?. [Day 3 (=SPECTROPHOTOMETRY: 1/5 DILUTION -> 100ul)]
Transfer 100ul in 4 black plates with transparent bottom
?. [Day 3 (=SPECTROPHOTOMETRY: 1/5 DILUTION -> 100ul)]
Procedure Details:  AB1Plate Type96 WELL PLATE2Eject plate on completion3Set TemperatureSetpoint 25°C4Preheat before moving to next step5ShakeFast, 0:10 (MM:SS)6ReadCell Density7Absorbance Endpoint8Full Plate9Wavelengths:&nbsp;&nbsp;60010Read Speed: Normal,&nbsp;&nbsp;Delay: 100 msec,&nbsp;&nbsp;Measurements/Data Point: 811ReadFluorescence Endpoint12Full Plate13Filter Set 114&nbsp;&nbsp;&nbsp;&nbsp;Excitation: 395/20,0,&nbsp;&nbsp;Emission: 508/20,015&nbsp;&nbsp;&nbsp;&nbsp;Optics: Top,&nbsp;&nbsp;Gain: 5016Filter Set 217&nbsp;&nbsp;&nbsp;&nbsp;Excitation: 395/20,0,&nbsp;&nbsp;Emission: 508/20,018&nbsp;&nbsp;&nbsp;&nbsp;Optics: Top,&nbsp;&nbsp;Gain: 7519Filter Set 320&nbsp;&nbsp;&nbsp;&nbsp;Excitation: 395/20,0,&nbsp;&nbsp;Emission: 508/20,021&nbsp;&nbsp;&nbsp;&nbsp;Optics: Top,&nbsp;&nbsp;Gain: 10022Read Speed: Normal,&nbsp;&nbsp;Delay: 100 msec,&nbsp;&nbsp;Measurements/Data Point: 1023Read Height: 8 mm
AB1Plate Type96 WELL PLATE2Eject plate on completion3Set TemperatureSetpoint 25°C4Preheat before moving to next step5ShakeFast, 0:10 (MM:SS)6ReadCell Density7Absorbance Endpoint8Full Plate9Wavelengths:&nbsp;&nbsp;60010Read Speed: Normal,&nbsp;&nbsp;Delay: 100 msec,&nbsp;&nbsp;Measurements/Data Point: 811ReadFluorescence Endpoint12Full Plate13Filter Set 114&nbsp;&nbsp;&nbsp;&nbsp;Excitation: 395/20,0,&nbsp;&nbsp;Emission: 508/20,015&nbsp;&nbsp;&nbsp;&nbsp;Optics: Top,&nbsp;&nbsp;Gain: 5016Filter Set 217&nbsp;&nbsp;&nbsp;&nbsp;Excitation: 395/20,0,&nbsp;&nbsp;Emission: 508/20,018&nbsp;&nbsp;&nbsp;&nbsp;Optics: Top,&nbsp;&nbsp;Gain: 7519Filter Set 320&nbsp;&nbsp;&nbsp;&nbsp;Excitation: 395/20,0,&nbsp;&nbsp;Emission: 508/20,021&nbsp;&nbsp;&nbsp;&nbsp;Optics: Top,&nbsp;&nbsp;Gain: 10022Read Speed: Normal,&nbsp;&nbsp;Delay: 100 msec,&nbsp;&nbsp;Measurements/Data Point: 1023Read Height: 8 mm