IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 3.1
- 7ml sterile tubes x4
- 1.5ml Eppendorf tube x1
- Room temperature 25oC
- Gilson Pipettes
- E.coli BL21; culture containing part :pTet-GFP
- LB medium
- Ampicillin stock (50 mg/ml)
- GFP Standard Solution
Innoculation of Media
- Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin
- 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)
- Well-plate x1
- Gilson pipettes 1000 and 200
- Eppendorf tubes
- GFP standard solution
Preparation of diluted GFP standard solution
- 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)
- Place the tube on ice till it is ready to be used.
- Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. (Follow the schematic as shown)
- Three wells to be filled with 200µl of media to measure the absorbance background.
- Standard GFP solution added as a positive control.
- Set counting times for each well according to schematic.
- Will the controls will be tested each time at each counting time ?
- 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)