http://2008.igem.org/wiki/index.php?title=Team:Caltech/Protocols/H2O2_Production_Assay&feed=atom&action=history
Team:Caltech/Protocols/H2O2 Production Assay - Revision history
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Revision history for this page on the wiki
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http://2008.igem.org/wiki/index.php?title=Team:Caltech/Protocols/H2O2_Production_Assay&diff=98608&oldid=prev
Cbeisel at 00:13, 30 October 2008
2008-10-30T00:13:33Z
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Grow an overnight culture of JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076 K137076] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2622 luxR] (negative control) on pSB1A2 in SOC + Amp.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Grow an overnight culture of JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076 K137076] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2622 luxR] (negative control) on pSB1A2 in SOC + Amp.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># In the morning, dilute culture 1:100 into 5 ml SOC +Amp +IPTG in triplicate.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># In the morning, dilute culture 1:100 into 5 ml SOC +Amp +IPTG in triplicate.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Grow <del class="diffchange diffchange-inline">culures </del>to an OD600 of ~0.8, then [[Team:Caltech/Protocols/Measuring_H2O2|assay supernatant]] for hydrogen peroxide approximately every 30 minutes.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Grow <ins class="diffchange diffchange-inline">cultures </ins>to an OD600 of ~0.8, then [[Team:Caltech/Protocols/Measuring_H2O2|assay supernatant]] for hydrogen peroxide approximately every 30 minutes.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># 1hr after reaching an OD600 of ~0.8, induce cultures with 10 nM AHL.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># 1hr after reaching an OD600 of ~0.8, induce cultures with 10 nM AHL.</div></td></tr>
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Cbeisel
http://2008.igem.org/wiki/index.php?title=Team:Caltech/Protocols/H2O2_Production_Assay&diff=57673&oldid=prev
Jkm at 22:06, 17 October 2008
2008-10-17T22:06:14Z
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Grow an overnight culture of JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076 K137076] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2622 luxR] (negative control) on pSB1A2 in SOC + Amp.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Grow an overnight culture of JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076 K137076] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2622 luxR] (negative control) on pSB1A2 in SOC + Amp.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># In the morning, dilute culture 1:100 into 5 ml SOC +Amp +IPTG in triplicate.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># In the morning, dilute culture 1:100 into 5 ml SOC +Amp +IPTG in triplicate.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Grow culures to an OD600 of ~0.8, then assay supernatant for hydrogen peroxide approximately every 30 minutes.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Grow culures to an OD600 of ~0.8, then <ins class="diffchange diffchange-inline">[[Team:Caltech/Protocols/Measuring_H2O2|</ins>assay supernatant<ins class="diffchange diffchange-inline">]] </ins>for hydrogen peroxide approximately every 30 minutes.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># 1hr after reaching an OD600 of ~0.8, induce cultures with 10 nM AHL.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># 1hr after reaching an OD600 of ~0.8, induce cultures with 10 nM AHL.</div></td></tr>
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Jkm
http://2008.igem.org/wiki/index.php?title=Team:Caltech/Protocols/H2O2_Production_Assay&diff=57671&oldid=prev
Jkm at 22:05, 17 October 2008
2008-10-17T22:05:25Z
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<td colspan='2' style="background-color: white; color:black;">Revision as of 22:05, 17 October 2008</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">An </del>overnight culture of JI377 transformed with <del class="diffchange diffchange-inline">luxR+GFP </del>or luxR (negative control) on pSB1A2 in SOC +Amp <del class="diffchange diffchange-inline">was back diluted </del>1:100 into 5 ml SOC +Amp +IPTG in triplicate. <del class="diffchange diffchange-inline">The cultures were grown </del>to an OD600 of ~0.8, <del class="diffchange diffchange-inline">at which point the </del>supernatant <del class="diffchange diffchange-inline">was assayed </del>for hydrogen peroxide approximately every 30 minutes. 1hr after reaching an OD600 of ~0.8, <del class="diffchange diffchange-inline">the </del>cultures <del class="diffchange diffchange-inline">were induced </del>with 10 nM AHL.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Grow an </ins>overnight culture of JI377 transformed with <ins class="diffchange diffchange-inline">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076 K137076] </ins>or <ins class="diffchange diffchange-inline">[http://partsregistry.org/wiki/index.php?title=Part:BBa_F2622 </ins>luxR<ins class="diffchange diffchange-inline">] </ins>(negative control) on pSB1A2 in SOC + Amp<ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># In the morning, dilute culture </ins>1:100 into 5 ml SOC +Amp +IPTG in triplicate.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Grow culures </ins>to an OD600 of ~0.8, <ins class="diffchange diffchange-inline">then assay </ins>supernatant for hydrogen peroxide approximately every 30 minutes.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># </ins>1hr after reaching an OD600 of ~0.8, <ins class="diffchange diffchange-inline">induce </ins>cultures with 10 nM AHL.</div></td></tr>
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Jkm
http://2008.igem.org/wiki/index.php?title=Team:Caltech/Protocols/H2O2_Production_Assay&diff=57629&oldid=prev
Tischerd at 19:40, 17 October 2008
2008-10-17T19:40:06Z
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<td colspan='2' style="background-color: white; color:black;">Revision as of 19:40, 17 October 2008</td>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><font face="verdana" style="color:#CC3300"><del class="diffchange diffchange-inline">Measuring </del>H<sub>2</sub>O<sub>2</sub> <del class="diffchange diffchange-inline">Concentration</del></font></div></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><font face="verdana" style="color:#CC3300">H<sub>2</sub>O<sub>2</sub> <ins class="diffchange diffchange-inline">Production Assay</ins></font></div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>__NOTOC__</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>__NOTOC__</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Approximately 100 uL </del>of <del class="diffchange diffchange-inline">cell culture was spun down. A colorimetric assay was performed using the PeroXOquant™ Quantitative Peroxide Assay Kit </del>(<del class="diffchange diffchange-inline">Pierce</del>) <del class="diffchange diffchange-inline">as per the manufacture’s instructions </del>in <del class="diffchange diffchange-inline">a 96 well plate. After incubating the reaction at room temperature for at least 20 minutes, it </del>was <del class="diffchange diffchange-inline">read on a plate reader (Spectramax M2) at 595nm</del>. The <del class="diffchange diffchange-inline">concentration </del>of hydrogen peroxide <del class="diffchange diffchange-inline">was calculated by reference to a standard (100uM, 33</del>.<del class="diffchange diffchange-inline">3uM, 11</del>.<del class="diffchange diffchange-inline">1 uM</del>, <del class="diffchange diffchange-inline">3.7 uM and 0.0 uM H2O2) made in </del>the <del class="diffchange diffchange-inline">corresponding media. Culture supernatants </del>were <del class="diffchange diffchange-inline">diluted as appropriate to bring measurements into the assay’s linear range</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">An overnight culture </ins>of <ins class="diffchange diffchange-inline">JI377 transformed with luxR+GFP or luxR </ins>(<ins class="diffchange diffchange-inline">negative control</ins>) <ins class="diffchange diffchange-inline">on pSB1A2 </ins>in <ins class="diffchange diffchange-inline">SOC +Amp </ins>was <ins class="diffchange diffchange-inline">back diluted 1:100 into 5 ml SOC +Amp +IPTG in triplicate</ins>. The <ins class="diffchange diffchange-inline">cultures were grown to an OD600 </ins>of <ins class="diffchange diffchange-inline">~0.8, at which point the supernatant was assayed for </ins>hydrogen peroxide <ins class="diffchange diffchange-inline">approximately every 30 minutes</ins>. <ins class="diffchange diffchange-inline">1hr after reaching an OD600 of ~0</ins>.<ins class="diffchange diffchange-inline">8</ins>, the <ins class="diffchange diffchange-inline">cultures </ins>were <ins class="diffchange diffchange-inline">induced with 10 nM AHL</ins>.</div></td></tr>
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Tischerd
http://2008.igem.org/wiki/index.php?title=Team:Caltech/Protocols/H2O2_Production_Assay&diff=57626&oldid=prev
Tischerd at 19:37, 17 October 2008
2008-10-17T19:37:11Z
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Insert protocol here</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">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.</ins></div></td></tr>
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Tischerd
http://2008.igem.org/wiki/index.php?title=Team:Caltech/Protocols/H2O2_Production_Assay&diff=43533&oldid=prev
Jkm at 20:37, 8 September 2008
2008-09-08T20:37:27Z
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><font face="verdana" style="color:#<del class="diffchange diffchange-inline">BB4400</del>">Measuring H<sub>2</sub>O<sub>2</sub> Concentration</font></div></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><font face="verdana" style="color:#<ins class="diffchange diffchange-inline">CC3300</ins>">Measuring H<sub>2</sub>O<sub>2</sub> Concentration</font></div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>__NOTOC__</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>__NOTOC__</div></td></tr>
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Jkm
http://2008.igem.org/wiki/index.php?title=Team:Caltech/Protocols/H2O2_Production_Assay&diff=43507&oldid=prev
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2008-09-08T18:36:59Z
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