IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 3.1

Equipment

 * 7ml sterile tubes x4
 * 1.5ml Eppendorf tube x1
 * Room temperature 25oC
 * Gilson Pipettes

Reagents

 * E.coli BL21; culture containing part :pTet-GFP
 * LB medium
 * Ampicillin stock (50 mg/ml)
 * GFP Standard Solution

Innoculation of Media
 * 1) Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin
 * 2) Incubate at 37°C for overnight in a shaker. (This is to get a good stock of cells for use in the experiment. After the overnight culture the cells will be in stationary phase)

Equipment

 * Well-plate x1
 * Fluorometer
 * Gilson pipettes 1000 and 200
 * Eppendorf tubes

Reagents

 * ddH2O
 * GFP standard solution

Preparation of diluted GFP standard solution
 * 1) Add 995ul of ultra pure water an eppendorf tube, together with 5ul of undiluted GFP standard solution and mix. (This gives a 200x dilution to be used as a positive control)
 * 2) Place the tube on ice till it is ready to be used.

Loading Plate
 * 1) Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. (Follow the schematic as shown)
 * 2) Three wells to be filled with 200µl of media to measure the absorbance background.
 * 3) Standard GFP solution added as a positive control.
 * 4) Set counting times for each well according to schematic.
 * 5) Will the controls will be tested each time at each counting time ?
 * 6) Remove lid and measure in the flourometer for 1 hour at 5 minute intervals.
 * (Fluorescence measurements - 488 nm excitation filter, 525 nm emission filter, vriable counting time, CW lamp energy 5000 units)

Schematic