TITLE: GFP-RFP plate reader assay
AUTHORS: Sebastiaan Kuiper

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Fluorescence measurements of Green fluorescent protein (GFP) and/or Red fluorescent protein (RFP) in </span><span style = "font-style:italic;">Escherichia coli.</span></div></div>

[STEPS]
?. [media preparations:]
Prepare the following media and autoclave them according to standard procedures.     M9TG Media:  ABC1  Reagent    Amount to add for 1L  Final concentration (1×)2  M9 salts    1x    1x  3  Tryptone    10 g    10 g/L  4  Glycerol    5 g    5 g/L      PBS buffer:  ABC1  Reagent    Amount to add for 1L  Final concentration (1×)2  NaCl    8 g    137 mM  3  KCl    0.2 g    2.7 mM  4  Na2HPO4    1.44 g    10 mM  5  KH2PO4    0.24 g    1.8 mM
ABC1  Reagent    Amount to add for 1L  Final concentration (1×)2  M9 salts    1x    1x  3  Tryptone    10 g    10 g/L  4  Glycerol    5 g    5 g/L
ABC1  Reagent    Amount to add for 1L  Final concentration (1×)2  NaCl    8 g    137 mM  3  KCl    0.2 g    2.7 mM  4  Na2HPO4    1.44 g    10 mM  5  KH2PO4    0.24 g    1.8 mM
?. [Plate reader protocol:]
Set the following variables for the BioTek Synergy MX Microplate Reader:-    Set Temperature: 37°CPreheat before moving to next step-    Shake: Medium, 0:30 (MM:SS)-    Read OD600:        Absorbance Endpoint of full plate        Wavelengths:600        Read Speed: Normal,Delay: 100 msec, Measurements/Data Point: 8-    Read GFP:     Fluorescence Endpoint of full plate      -   Filter Set 1:         Excitation: 485/9,0,Emission: 512/9,0         Optics: Bottom,Gain: 50    -    Filter Set 2:         Excitation: 485/9,0,Emission: 512/9,0         Optics: Bottom,Gain: 75    -    Filter Set 3         Excitation: 485/9,0,Emission: 512/9,0         Optics: Bottom,Gain: 100    Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 10-    Read RFP:     Fluorescence Endpoint of full plate    -    Filter Set 1:         Excitation: 555/9,0,Emission: 585/9,0         Optics: Bottom,Gain: 100    -    Filter Set 2:         Excitation: 555/9,0,Emission: 585/9,0         Optics: Bottom,Gain: 150    Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 10
?. [Fluorescence analysis:]
Grow desired bacteria overnight in a 50 ml falcon tub, containing  and grown cultures @, 250 RPM overnight.
[M9TG media]
37 °C
Luria-Bertani media is also possible to use, but cells must be washed with PBS buffer prior to plate reader experiment because of the high amount of fluorescence from the yeast extract:    After overnight incubation, spin cells down at 4700 x G, for 5 min, discard the supernatant and resuspend in 10 ml PBS.     Repeat prior steps and resuspension again in PBS.
?. [Fluorescence analysis:]
Load  in the wells of the 96-well plate (with clear bottom and black sides) and add
[PBS buffer]
[of overnight culture]
When cell have been grown overnight in LB media, washing steps are require before continuing!  (see step 3)
Add as s control a sample with    and  .
?. [Fluorescence analysis:]
Start plate reader protocol
?. [Analyses]
Retrieve the data from the computer, correct for the measurements for the OD600 and subtract the autofluorescence from the control sample of all the other samples.