Initial Testing for DNA constructs In Vivo: Difference between revisions
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Peixuan Pey (talk | contribs) (New page: ==Protocol== * Status - <font>Completed</font> ===Day 1=== ====Equipment==== *7ml sterile tubes x4 *1.5ml Eppendorf tube x1 *Room temperature 25<sup>o</sup>C *Gilson Pipettes ====Reagent...) |
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Revision as of 05:40, 12 September 2007
Protocol
- Status - Completed
Day 1
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
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)
Day 2
Equipment
- Well-plate x1
- Fluorometer
- Gilson pipettes 1000 and 200
- Eppendorf tubes
Reagents
- ddH2O
- 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.
Loading Plate
- 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.
- 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)
- Repeat the measurement a further two times straight after each other (This is to test the variability of the machine)
Schematic
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