Imperial/Wet Lab/Protocols/ID1.1
From 2007.igem.org
Wet Lab: Protocols: Initial Testing for DNA Constructs in vivo
Aims
- To test to see if the DNA construct from the registry are viable. This is done in vivo.
- The constructs are pTet-LuxR-pLux-GFP.
Day 1
Equipment
- 7ml sterile tubes x4
- 1.5ml Eppendorf tube x1
- 37°C incubator
- Gilson Pipettes p1000 p200 p20
Reagents
- E.coli BL21; culture containing pTet-LuxR-pLux-GFP
- LB medium
- Ampicillin stock (50 mg/ml)
- AHL stock
Innoculation of Media
- Inoculate 10ul of stored parts 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.)
Preparation of culture for AHL induction and GFP measurement
- From the stock culture of transformed E.coli, dilute the culture so that the OD=0.1.
Preparation of diluted series of AHL
http://partsregistry.org/wiki/images/4/4c/F2620-TF-Small.png
(Taken off [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]. Results show required dilution of AHL solution.)
- Add 6µl of AHL stock solution to 600µl of cell samples, to obtain a final concentration of 1uM.
Day 2
Equipment
- 37°C incubator
- Fluorometer + PC
- Gilson pipettes 1000 and 200
- 1 Fluorometer plate (black)
- Eppendorf Tubes
- Stop watch
Reagents
- LB medium
- E.coli culture with transformed plasmid
- 10μM AHL stock
- 100μM AHL stock
- GFP stock solution
- ddH2O
Protocol
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
1. 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.
- Three further wells to be filled with 200 µl of (media + empty-vector culture) to measure fluorescent background.
- Standard GFP solution added as a positive control.
2. Add Xµl of stock concentration of AHL to the respective wells as shown. 3. Incubate it at 37oC for 4 hours. 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)
3. 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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