IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.2: Difference between revisions
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''After 4hrs in the shaker'': | |||
*Add a 200uL sample from each eppendorf tube to the 96 well plate. | |||
*Do this for 8 repeats following suggested patterning (see above) | |||
**NOTE: Since all repeats are made from the same culture, it is enuogh to do 4 repeats - thus one plate can be used for 2 different tests. (Pipetting errors will be ruled out since absorbance is measured later and can then be considered when processing the data.) | |||
*Add 4 x 200uL of growth medium to a well to act as a control. | |||
*Take the plate and eppendorfs to BCHEM | |||
<showhide> | |||
*Add 995ul of ultra pure water to the eppendorf, together with 5ul of undiluted GFP standard solution and mix __HIDER__ | |||
<hide> | |||
:<font color=green>A 200 x dilution of GFP is made. <br> | |||
:The undiluted GFP is in the BCHM Level 6 Cold Room on our shelf. It is in a small grey plastic box. Pippettes, tips and ultrapure water are located on a shelf above the electroporation machine in the plate reader room.</font> | |||
</hide> | |||
</showhide> | |||
*Add 4 x 200uL of the 200x diluted GFP standard solution to the wells following the suggested patterning | |||
*Take a reading | |||
**Take the plate to the plate reader room | |||
<showhide> | |||
:*Use the Victor3 to measure flourescence and absorbance __HIDER__ | |||
<hide> | |||
::<font color=green>Use the preprogrammed Assay under the 'Students' folder called GFP + Abs490</font> | |||
</hide> | |||
</showhide> | |||
<showhide> | |||
*Repeat the measurment a further two times straight after each other __HIDER__ | |||
<hide> | |||
:<font color=green>This to assess the variability of the machine</font> | |||
</hide> | |||
</showhide> | |||
<showhide> | |||
*Save data file from computer. __HIDER__ | |||
<hide> | |||
:<font color=green>You'll need a memory stick to save the information to. Unfortunatley the computer isn't networked</font> | |||
</hide> | |||
</showhide> | |||
*Copy and paste the data into a [http://openwetware.org/images/a/a2/T9002_Data_Spreadsheet.xls T9002/J37016/J37020 Data Spreadsheet] | |||
Revision as of 08:36, 14 August 2007
Protocol
Day 1
Equipment
- 7ml tubes x4
- Incubator 37oC
- Gilson Pipettes
Reagents
- E.coli DH5α 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 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)
Day 2
Equipment
- Spectrophotometer
- Stripettes
- Well-plate x1
- Plate Centrifuge
- Stop watch
Reagents
- LB medium
- Ampicillin stock (50 mg/ml)
- GFP Standard Solution
- AHL stock
Innoculation of Media (cont'd)
- In the morning, prewarm 20 mL LB Amp medium in the 37°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)
- Return LB to 37°C waterbath. (This returns cells to exponential phase from stationary phase)
- Incubate new culture at 37°C
Preparation of diluted series of AHL
- Dilution series of AHL [1000 μM, 100 μM, 10 μM, 5 μM]
- Vortex new T9002/J37016/J37020 culture.
- To start AHL incubation:
- Label each tube with AHL concentration
- Put 20ul of the AHL into 11 seperate 5ml white capped tubes
- Add appropriate amount of T9002 samples as per the table below
- Record time of inoculation in report sheet.
- Vortex each tube
<showhide>
- Incubate all 5mL tubes in a 37°C shaker for 4 hours so GFP expression can reach steady state __HIDER__
<hide>
- Do not pipette the samples into the 96 well plate yet - because if the 96 well plate is put in the shaker, cross-contamination between the well is very likely to happen.
</hide></showhide>
Loading Plate
- Follow the schematic for the plate adding everything but the DNA sample.
- Place the top on the plate and place in the incubator. Leave for a few minutes to heat to 30oC
- Remove from incubator and centrifuge for 1 minute
- Remove lid and Measure in the flourometer.
- Then to begin the reaction add .... purified DNA sample.
- Place lid back on and place back in the incubator at 30oC
Schematic
|
After 4hrs in the shaker:
- Add a 200uL sample from each eppendorf tube to the 96 well plate.
- Do this for 8 repeats following suggested patterning (see above)
- NOTE: Since all repeats are made from the same culture, it is enuogh to do 4 repeats - thus one plate can be used for 2 different tests. (Pipetting errors will be ruled out since absorbance is measured later and can then be considered when processing the data.)
- Add 4 x 200uL of growth medium to a well to act as a control.
- Take the plate and eppendorfs to BCHEM
<showhide>
- Add 995ul of ultra pure water to the eppendorf, together with 5ul of undiluted GFP standard solution and mix __HIDER__
<hide>
- A 200 x dilution of GFP is made.
- The undiluted GFP is in the BCHM Level 6 Cold Room on our shelf. It is in a small grey plastic box. Pippettes, tips and ultrapure water are located on a shelf above the electroporation machine in the plate reader room.
</hide> </showhide>
- Add 4 x 200uL of the 200x diluted GFP standard solution to the wells following the suggested patterning
- Take a reading
- Take the plate to the plate reader room
<showhide>
- Use the Victor3 to measure flourescence and absorbance __HIDER__
<hide>
- Use the preprogrammed Assay under the 'Students' folder called GFP + Abs490
</hide> </showhide> <showhide>
- Repeat the measurment a further two times straight after each other __HIDER__
<hide>
- This to assess the variability of the machine
</hide> </showhide> <showhide>
- Save data file from computer. __HIDER__
<hide>
- You'll need a memory stick to save the information to. Unfortunatley the computer isn't networked
</hide> </showhide>
- Copy and paste the data into a T9002/J37016/J37020 Data Spreadsheet
Equipment
- Incubator 30oC
- Fluorometer
- Spectrometer
- Plate x1
- Plate Centrifuge
- 6x1.5ml tubes
- Gilson Pipettes
- Stop watch
Day 3:
Make 5 of 550 ml LB medium with 550 µl ampicillin in sterile conical flask Innoculate in 5 cultures into the the conical flasks Leave overnight at 37°C with shaking
Day 4:
Prepare a water bath at 30°C [should we use a shaking incubator?] Aliquot the 550 ml cultures into sterile tubes, 25 ml per tube Add 53.3075 ng AHL each time to increase [AHL] in steps of 10nM Begin the timer when AHL is added At 5 minute intervals for the first 40 minutes, and 10 minute intervals thereafter: Sample a 1 ml of the reaction mixture Immediately chill in ice (to slow the rate of reaction) Centrifuge at maximum speed for 30 seconds to pellet the cells Remove the supernatant and resuspend the pellet in 1ml distilled water Put in a spectrometer and excite it at 501nm and detect at 511nm [Note: Use solution at 0 minutes as a blank, assume no fluorescence is present prior to activation] These measurements should be repeated with the other 4 cultures of bacteria