Team:BIOTEC Dresden/ProtocolsAHL assay

From 2010.igem.org

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Please find the protocol <a href="http://igem.org/wiki/images/7/75/Protocol_AHLassay.pdf"> here.</a
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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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Revision as of 01:16, 28 October 2010

Please find the protocol here.
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