Initial Testing for DNA constructs of CBD In Vivo

Aims
 * To test to see if the DNA constructs from the registry are viable. This is done in vivo.
 * The constructs are pTet-GFP, pT7-GFP and pcI-GFP.

Equipment

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

Reagents

 * E.coli BL21; culture containing parts :pTet-GFP, pT7-GFP, pcI-GFP
 * LB medium


 * Ampicillin stock (50 mg/ml)
 * GFP Standard Solution - This concentration was unknown, however because we only wanted it as a positive control to show there was fluorescence and that the flurometer could measure it

Protocol
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

Protocol
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) Remove lid and measure in the flourometer.
 * (Fluorescence measurements - 488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units)


 * 1) Repeat the measurement a further two times straight after each other (This is to test the variability of the machine)

Schematic