IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.2: Difference between revisions
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====Reagents==== | ====Reagents==== | ||
*LB medium | *LB medium | ||
*E.coli culture with transformed plasmid | |||
*Ampicillin stock (50 mg/ml) | *Ampicillin stock (50 mg/ml) | ||
*10μM AHL stock | *10μM AHL stock | ||
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<br> | <br> | ||
From the stock culture of transformed E.coli, dilute the culture so that the OD=0.1. | |||
'''Loading Plate''' | '''Loading Plate''' |
Revision as of 05:46, 23 August 2007
Protocol(Status: ???)
- define status: Work in Progress / Feedbacks Required / Validated (other tags ?)
Day 1
Equipment
- 7ml sterile tubes x4
- 1.5ml Eppendorf tube x1
- Incubator 37oC
- Gilson Pipettes
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 diluted series of AHL
http://parts.mit.edu/registry/images/4/4c/F2620-TF-Small.png
(Taken off BBa_F2620. Results show required dilution of AHL solution)
- Add 2µl of AHL stock solution to 200µl of cell samples, to a final concentratino of 1uM.
Day 2
Equipment
- Water bath @ 37°C
- Stripettes
- Well-plate x1
- Stop watch
Reagents
- LB medium
- E.coli culture with transformed plasmid
- Ampicillin stock (50 mg/ml)
- 10μM AHL stock
- 100μM AHL stock
- GFP stock solution
- ddH2O
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)
From the stock culture of transformed E.coli, dilute the culture so that the OD=0.1.
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.
- Three further wells to be filled with 200 µl of (media + empty-vector culture) to measure fluorescent background. (IS THIS POSSIBLE??!?!)
- Standard GFP solution added as a positive control.
- Add Xµl of stock concentration of AHL to the respective wells as shown.
- Incubate it at 37oC for 4 hours.
- 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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Collecting Data
- After 5 minutes of incubation measure the fluorescence by repeating procedure 3-5 above.
- Repeat measurements after every 5 minutes until the fluorescence is constant
- Before every measurement in the fluorometer spin the plates in a plate centrifuge
- Save data file from computer
- Copy and paste the data into an Excel spreadsheet for data analysis