IGEM:IMPERIAL/2006/Protocols/T9002: Difference between revisions
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*Inoculate a culture from 10ul of stored T9002 in 2ml LB/M9 growth medium containing 50ug/ml Ampicilin. | *Inoculate a culture from 10ul of stored T9002 in 2ml LB/M9 growth medium containing 50ug/ml Ampicilin. | ||
*Incubate at 37oC for overnight in a shaker. | *Incubate at 37oC for overnight in a shaker. | ||
*Following day, | *Following day, prewarm LB/M9 to 37oC by placing in the 37oC incubator | ||
*Measure and record OD600_1 in report sheet | |||
*Inoculate a 8ml fresh culture from the o/n to bring back the OD600 to 0.1, use prewarmed LB/M9 + Ampicilin. | *Inoculate a 8ml fresh culture from the o/n to bring back the OD600 to 0.1, use prewarmed LB/M9 + Ampicilin. | ||
** Volume used to inoculate new culture = (0.1/OD600_1)*8ml | ** Volume used to inoculate new culture = (0.1/OD600_1)*8ml | ||
*Return LB/M9 to incubator | |||
*Incubate new culture at 37oC for 2 hours in a shaker - '''<font color = green>This returns cells to exponential phase</font>''' | *Incubate new culture at 37oC for 2 hours in a shaker - '''<font color = green>This returns cells to exponential phase</font>''' | ||
*Measure and record the OD600_2 in report sheet | *Measure and record the OD600_2 in report sheet | ||
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*Add 4 x 200uL of growth medium to a well to act as a control. | *Add 4 x 200uL of growth medium to a well to act as a control. | ||
*Incubate the 96 well palte in a 37oC shaker for 4 hours so GFP expression can reach steady state. | *Incubate the 96 well palte in a 37oC shaker for 4 hours so GFP expression can reach steady state. | ||
*After the 4 hours add 4 x 200uL of 200x diluted GFP standard solution <font color=red> why 4 ?</font> <font color=green>to account for variability in readings, if necessary can just read the same well multiple times</font> | *After the 4 hours add 4 x 200uL of 200x diluted GFP standard solution <font color=red> why 4 ?</font> <font color=green>to account for variability in readings/pipetting, if necessary can just read the same well multiple times</font> | ||
*Take 3 readings with a 20 minute interval | *Take 3 readings with a 20 minute interval | ||
*Taking readings | *Taking readings | ||
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'''Last Updated:''' [[User:TomH|Tom]] | '''Last Updated:''' [[User:TomH|Tom]] 14:53, 14 August 2006 (BST) | ||
Revision as of 06:53, 14 August 2006
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 transfer function 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
- 12 Ependorf Tubes
- Gilson Pippettes
- 37oC Shaker
- T9002 Report Sheet
- Materials
- Dilution series of AHL
- GFP Standard Solution
- E.coli Growth Medium w/Ampicilin (LB/M9)
- E.coli DH5a Culture Containing T9002
Protocol
- Inoculate a culture from 10ul of stored T9002 in 2ml LB/M9 growth medium containing 50ug/ml Ampicilin.
- Incubate at 37oC for overnight in a shaker.
- Following day, prewarm LB/M9 to 37oC by placing in the 37oC incubator
- Measure and record OD600_1 in report sheet
- Inoculate a 8ml fresh culture from the o/n to bring back the OD600 to 0.1, use prewarmed LB/M9 + Ampicilin.
- Volume used to inoculate new culture = (0.1/OD600_1)*8ml
- Return LB/M9 to incubator
- Incubate new culture at 37oC for 2 hours in a shaker - This returns cells to exponential phase
- Measure and record the OD600_2 in report sheet
- Dilute again for an OD of 0.1 in a new culture of 15ml of a prewarmed LB/M9 + Ampicilin.
- Volume used to inoculate new culture = (0.1/OD600_2)*15ml
- Vortex new T9002 culture.
- Put 990ul of the culture into 11 seperate 1.5ml eppendorf tubes to start AHL incubation:
- Label each tube with AHL concentration
- Add AHL from stock
- Record time of AHL inoculation in report sheet.
- Vortex each tube
|
we should optimize usage of the 96 plate, more samples needed maybe from different colonies maybe better to include a control of medium with non-flourescing cells also, see experimental data for reasons
- Get 200uL samples from each eppendorf tubes to the 96 well plate.
- Do this for 4 repeats following suggested patterning (see above)
- Add 4 x 200uL of growth medium to a well to act as a control.
- Incubate the 96 well palte in a 37oC shaker for 4 hours so GFP expression can reach steady state.
- After the 4 hours add 4 x 200uL of 200x diluted GFP standard solution why 4 ? to account for variability in readings/pipetting, if necessary can just read the same well multiple times
- Take 3 readings with a 20 minute interval
- Taking readings
- Take the plate from shaker
- Use the Victor3 to measure flourescence and absorbance
- Return the plate to the 37C water bath VR: Don't know if standard GFP will be that stable if incubated at 37C for 1h Agreed, however we only need one time point for the callibration. The rest can be ignored.
- Save data file from computer.
Any questions, see Tom.
Last Updated: Tom 14:53, 14 August 2006 (BST)
Many thanks to Drew Endy and Barry Canton from MIT for providing the protocol on which this is based.
Potential Issues
- We should make a frozen stock of T9002. Maybe 4-5 different colonies from a plate.
- M9 or LB?
- Need information from J37015 as to whether AHL production can be accomplished in M9
- What is the GFP standard solution? Do we know its concentration? How stable is that? half life?
- JS: Please can you write a protocol that will decouple other experiments with this one in case we want to assay an unknown concentration of AHL. Thanks. (i.e. take an unknown concentration of AHL of x amount into the T9002....measure flourescence...etc)