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From 2010.igem.org

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