Team:Caltech/Protocols/H2O2 Production Assay

(Difference between revisions)

Latest revision as of 00:13, 30 October 2008

iGEM 2008

Home

People

Project Details

Protocols

Completed Systems

Biosafety

Support

H₂O₂ Production Assay

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.
In the morning, dilute culture 1:100 into 5 ml SOC +Amp +IPTG in triplicate.
Grow cultures to an OD600 of ~0.8, then assay supernatant for hydrogen peroxide approximately every 30 minutes.
1hr after reaching an OD600 of ~0.8, induce cultures with 10 nM AHL.

@@ Line 3: / Line 3: @@
 <div style="font-size:18pt;">
-<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__
 <br>
-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.
+# 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.
+# In the morning, dilute culture 1:100 into 5 ml SOC +Amp +IPTG in triplicate.
+# Grow cultures to an OD600 of ~0.8, then [[Team:Caltech/Protocols/Measuring_H2O2|assay supernatant]] for hydrogen peroxide approximately every 30 minutes.
+# 1hr after reaching an OD600 of ~0.8, induce cultures with 10 nM AHL.
 }}