IGEM:IMPERIAL/2006/project/Oscillator/project browser/Test Sensing Predator Construct/TestingValidation
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| Super Parts | Predator Construct | |||
|---|---|---|---|---|
| Actual Part | Logo of the Part | |||
| Sub Parts | intermediate_part | intermediate_part | intermediate_part | intermediate_part |
- Inoculate a culture from a single bacterial colony in LB growth medium containing appropriate antibiotic.
- Incubate at 37oC for 15 hours in a shaker.
- Remove cultures from shaker and dilute by a factor of 10, by adding 1ml of culture into 9ml of fresh LB medium.
- Incubate at 37oC for 2 hours in a shaker - This returns cells to exponential phase
- We have a stock AHL solution of 1000uM
- Serial dilute to give final 1ml stock concentrations of 1000uM, 100uM, 10uM, 1uM, 100nM, 10nM, 1nM (check the -20oC Freezer as these may be already made up).
- Add the following to eight different labelled ependorf tubes (remember to add the AHL last):
| Sample (ul) | Stock Concentration | AHL (ul) | Final AHL Concentration |
|---|---|---|---|
| 990 | 1000uM | 10 | 10uM |
| 990 | 100uM | 10 | 1uM |
| 990 | 10uM | 10 | 100nM |
| 990 | 1uM | 10 | 10nM |
| 990 | 100nM | 10 | 1nM |
| 990 | 10nM | 10 | 0.1nM |
| 990 | 1nM | 10 | 0.01nM |
| 1000 | N/A | 0 | 0nM |
- Add 200uL of growth medium to a well to act as a control
- Vortex tubes for 1 second
- Incubate the culture in a 37oC incubator, taking readings every 30minutes
- Taking readings
- Prepare an ice bucket containing a pre-chilled 96 well plate - The ice is used to stop cell activity, therefore its imporant the plate stays cold
- Remove the tubes from the incubator
- Pippet 200ul samples from each tube into the 96 well plate inside the ice bucket
- Vortex the tubes for 1 second - As the cells aren't in the shaker, the vortex helps to airate the culture
- Return tubes to the oven
- Repeat for a total of 4 measurments
- Take 96 well plate in ice bucket to the SAF
- Record data from the fluorimeter
References
Tom Hinson
