Team:Caltech/Protocols/H2O2 Production Assay

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H₂O₂ Production Assay

An overnight culture of JI377 transformed with luxR+GFP or luxR (negative control) on pSB1A2 in SOC +Amp was back diluted 1:100 into 5 ml SOC +Amp +IPTG in triplicate. The cultures were grown to an OD600 of ~0.8, at which point the supernatant was assayed for hydrogen peroxide approximately every 30 minutes. 1hr after reaching an OD600 of ~0.8, the cultures were induced with 10 nM AHL.

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-<font face="verdana" style="color:#CC3300">Measuring H<sub>2</sub>O<sub>2</sub> Concentration</font></div>
+<font face="verdana" style="color:#CC3300">H<sub>2</sub>O<sub>2</sub> Production Assay</font></div>
 __NOTOC__
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-Approximately 100 uL of cell culture was spun down. A colorimetric assay was performed using the PeroXOquant™ Quantitative Peroxide Assay Kit (Pierce) as per the manufacture’s instructions in a 96 well plate. After incubating the reaction at room temperature for at least 20 minutes, it was read on a plate reader (Spectramax M2) at 595nm. The concentration of hydrogen peroxide was calculated by reference to a standard (100uM, 33.3uM, 11.1 uM, 3.7 uM and 0.0 uM H2O2) made in the corresponding media. Culture supernatants were diluted as appropriate to bring measurements into the assay’s linear range.
+An overnight culture of JI377 transformed with luxR+GFP or luxR (negative control) on pSB1A2 in SOC +Amp was back diluted 1:100 into 5 ml SOC +Amp +IPTG in triplicate. The cultures were grown to an OD600 of ~0.8, at which point the supernatant was assayed for hydrogen peroxide approximately every 30 minutes. 1hr after reaching an OD600 of ~0.8, the cultures were induced with 10 nM AHL.
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