IGEM:IMPERIAL/2006/Protocols/T9002: Difference between revisions
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**E.coli Culture Containing T9002 | **E.coli Culture Containing T9002 | ||
=== | ===Protocol=== | ||
*Inoculate a culture from 10ul of stored T9002 in 2ml LB/M9 growth medium containing 50mg/ml Ampicilin. | *Inoculate a culture from 10ul of stored T9002 in 2ml LB/M9 growth medium containing 50mg/ml Ampicilin. | ||
*Incubate at 37oC for overnight in a shaker. | *Incubate at 37oC for overnight in a shaker. |
Revision as of 05:31, 10 August 2006
comments from Vincent
VR: To describe your protocol:
- define motivation behing it + link to part used
- list equipment needed
- list different steps of the protocol - bullet points -
- discuss potential issues
Part T9002: Transfer Function Characterisation
Motivation
The motivation behind this test construct is to characterise the transfer function linking AHL input to GFP output.
This will allow us to construct a standard curve which will allow us to relate GFP output to an unknown AHL input, forming the basis of an AHL assay.
Materials & Equipment
- Equipment
- Wallac Victor 3 Multi-Well Fluorimeter
- 11 Ependorf Tubes
- Gilson Pippettes
- 37oC Shaker
- Materials
- AHL
- GFP Standard Solution
- E.coli Growth Medium w/Ampicilin (LB/M9)
- E.coli Culture Containing T9002
Protocol
- Inoculate a culture from 10ul of stored T9002 in 2ml LB/M9 growth medium containing 50mg/ml Ampicilin.
- Incubate at 37oC for overnight in a shaker.
- Measure and record OD
- Remove cultures from shaker and dilute so the culture is at an OD of 0.1 using prewarmed LB/M9.
- OD/0.1 x 2 = Total volume required for dilution
- Incubate at 37oC for 2 hours in a shaker - This returns cells to exponential phase
- Measure and record the OD
- Dilute again with prewarmed LB/M9 for an OD of 0.1.
- Add the following to 11 seperate ependorf tubes:
Sample (ul) | Stock Concentration | AHL (ul) | Final AHL Concentration |
---|---|---|---|
990 | 1000uM | 10 | 10uM |
990 | 100uM | 10 | 1uM |
990 | 10uM | 10 | 100nM |
990 | 5uM | 10 | 50nM |
990 | 1uM | 10 | 10nM |
990 | 500nM | 10 | 5nM |
990 | 100nM | 10 | 1nM |
990 | 50nM | 10 | 0.5nM |
990 | 10nM | 10 | 0.1nM |
990 | 1nM | 10 | 0.01nM |
1000 | N/A | 0 | 0nM |
- Incubate the ependorfs in a 37oC shaker for 4 hours
- After the 4 hours add 200uL samples from the ependorf tubes to the 96 well plate
- Do this for 5 repeats
- Add 200uL of growth medium to a well to act as a control
- Add 200uL of 200x diluted GFP standard solution
- Take 3 readings with a 20 minute interval
- Taking readings
- Take the plate from the water bath
- Use the Victor3 to measure flourescence and absorbance
- Return the plate to the water bath
Any questions, see Deepti or Tom.
Last Updated: Tom 12:03, 8 August 2006 (BST)
Many thanks to Drew Endy and Barry Canton from MIT for providing the protocol on which this is based.
Potential Issues
- What is the GFP standard solution? Do we know its concentration? How stable is that? half life?
- Growing all the five repeats together in one ependorf tube, can we get around this?
- Incuabtor that can shake with the 96 well plate?