IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.2: Difference between revisions
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: Standard GFP solution added as a positive control. | : Standard GFP solution added as a positive control. | ||
#Add 2µl of stock concentrations of AHL (10μM & 100μM) to the respective wells as shown. | |||
#Place the top on the plate and centrifuge for 1 minute | #Place the top on the plate and centrifuge for 1 minute | ||
#Remove lid and measure in the flourometer. | #Remove lid and measure in the flourometer. |
Revision as of 03:41, 15 August 2007
Protocol
Day 1
Equipment
- 7ml sterile tubes x4
- 1.5ml Eppendorf tube x1
- Incubator 30oC
- Gilson Pipettes
Reagents
- E.coli BL21; culture containing parts
- LB medium
- Ampicillin stock (50 mg/ml)
- GFP Standard Solution
- AHL stock
Innoculation of Media
- Inoculate 10ul of stored parts in individual 2ml LB medium containing 2ul of ampicillin
- Incubate at 30°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 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)
Day 2
Equipment
- Water bath @ 30°C
- Spectrophotometer
- Stripettes
- Well-plate x1
- Plate Centrifuge
- Stop watch
Reagents
- LB medium
- Ampicillin stock (50 mg/ml)
- Diluted GFP standard solution
- 10μM AHL stock
- 100μM AHL stock
Innoculation of Media (cont'd)
- In the morning, prewarm 20 mL LB Amp medium in the 30°C water bath.
- Measure and record OD600
- Inoculate a 10ml fresh culture from the o/n to bring back the OD600 to 0.1, use the prewarmed LB + Ampicilin in waterbath. (Using prewarmed LB prevents a temperature shock to the culture, which would increase lag time)
\frac{0.1}{\mbox{OD of culture}} \times \mbox{10 mL}
</amsmath>- OD of XXX culture (1st Measurement): _________
- Amount to dilute of XXX culture = ________ mL (amount of original culture to use)
- Amount of prewarmed LB with Ampicillin to use = ________ mL (16 mL - above result)
- This returns cells to exponential phase from stationary phase
- Return LB to 30°C waterbath.
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 2µl of stock concentrations of AHL (10μM & 100μM) to the respective wells as shown.
- Place the top on the plate and centrifuge for 1 minute
- 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)
- Place lid back on and place back in the incubator at 30oC
Schematic
|
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