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| - | | + | Please find the protocol <a href="https://static.igem.org/mediawiki/igem.org/7/75/Protocol_AHLassay.pdf"> here.</a |
| - | Preparing a 96-well plate for HHL assay
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| - | 1. Set-up
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| - | Column1: 180µl LB-medium + 20µl H2O
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| - | Column2: 180µl cells + 20µl H2O
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| - | Column3: 200µl cells in 0.01nM HHL
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| - | Column4: 200µl cells in 0.1nM HHL
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| - | Column5: 200µl cells in 1nM HHL
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| - | Column6: 200µl cells in 10nM HHL
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| - | Column7: 200µl cells in 50nM HHL
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| - | Column8: 200µl cells in 100nM HHL
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| - | Column9: 200µl cells in 200nM HHL
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| - | Column10: 200µl cells in 500nM HHL
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| - | Column11: 200µl cells in 1000nM HHL
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| - | Column12: 200µl cells in 2000nM HHL
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| - | 2. Pipetting scheme
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| - | 2.1. Stock solutions
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| - | Final con. / µM Stock used / µM Vol. of stock / µl Vol. of water / µl
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| - | 150 15000 12 1188
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| - | 15 150 120 1080
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| - | 1.5 15 120 1080
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| - | 0.15 1.5 120 1080
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| - | 0.015 0.15 120 1080
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| - | 2.2. 94-well plate
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| - | The volumes below are sufficient for pipetting four well plates at a time.
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| - | Final con. / nM Stock used / µM Vol. of stock / µl Vol. of water / µl
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| - | 0.01 0.015 4.8 715.2
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| - | 0.1 0.015 48 672
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| - | 1 0.15 48 672
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| - | 10 0.15 480 240
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| - | 50 1.5 240 480
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| - | 100 1.5 480 240
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| - | 200 15 96 624
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| - | 500 15 240 480
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| - | 1000 15 480 240
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| - | 2000 150 96 624
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| - | Pipet 20µl of the above concentrations into the corresponding wells and add 180µl of cell suspension right before the measurement of fluorescence.
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| - | 3. Tecan plate reader
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| - | Start the machine 20 minutes in advance and heat the chamber to 37°C. Put in the plate and start the measurement with the following setting:
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| - | • Measure OD at 612 nm
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| - | • Measure fluorescence with an excitation wavelength of 485nm and an emission wavelength of 485nm for both GFP and YFP
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| - | • Shake
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| - | • Repeat measurements every 5min for 3 hours
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| - | 4. Data processing
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| - | • Normalize the data by dividing all fluorescence data by the corresponding optical density
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| - | • Subtract the obtained value of the reference column (column 2) from the calculated relative fluorescence of the wells containing HHL
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| - | • Plot the fluorescence data over the time
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"
Please find the protocol here.
