Team:Calgary/Notebook/Protocols/escapersassay

From 2012.igem.org

(Difference between revisions)
Line 5: Line 5:
<p>The following assay is based on CFU in both Top 10 and glyA knockout of <i>E. coli</i>.</p>
<p>The following assay is based on CFU in both Top 10 and glyA knockout of <i>E. coli</i>.</p>
 +
<ol>
 +
<li>Prha-B0034-S7 was transformed into Top 10 and glyA knockout.</li>
 +
 +
<li>Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.</li>
 +
 +
<li>The cultures were then aliquoted into equal volumes and washed with the appropriate condition media.</li>
<ul>
<ul>
-
<li>The <i>glyA</i> auxotroph was grown in LB media overnight at 37<sup>o</sup>C with shaking.
+
<li>M9 minimal media with 0.2% glucose.</li>
-
<li>Cells were washed in M9 minimal media two times and spun in between at 4,000 RPM at 4<sup>o</sup>C for 10 min. each time.
+
<li>M9 minimal media with 0.2% rhamnose.</li>
-
<li> Finally, washed cells were subcultured into 5 mL of M9 minimal media supplemented with 1% (w/v) glucose and 50 ug/mL Kanamycin.
+
-
<li>Cells were grown at 37<sup>o</sup>C with shaking for 12 and 24 hours and OD<sub>600</sub> measurements were used to quantitate growth.
+
</ul>
</ul>
 +
 +
 +
<li>All the different cell lines (pRHA-B0034-S7 in Top 10, pRHA-B0034-S7 in gly knockout, gly knockout) were washed with the appropriate media and then plated at dilutions of 1:1000, 1:10000, 1:100000 at 0 hr, 4 hr, 8 hr, 24 hr.</li>
 +
 +
 +
</ol>
</html>
</html>
}}
}}

Revision as of 03:35, 27 October 2012

Hello! iGEM Calgary's wiki functions best with Javascript enabled, especially for mobile devices. We recommend that you enable Javascript on your device for the best wiki-viewing experience. Thanks!

Escaper assay for the rhamnose system

The following assay is based on CFU in both Top 10 and glyA knockout of E. coli.

  1. Prha-B0034-S7 was transformed into Top 10 and glyA knockout.
  2. Colonies were inoculated in LB with 2% glucose to make sure the system was turned off and the cells were growing.
  3. The cultures were then aliquoted into equal volumes and washed with the appropriate condition media.
    • M9 minimal media with 0.2% glucose.
    • M9 minimal media with 0.2% rhamnose.
  4. All the different cell lines (pRHA-B0034-S7 in Top 10, pRHA-B0034-S7 in gly knockout, gly knockout) were washed with the appropriate media and then plated at dilutions of 1:1000, 1:10000, 1:100000 at 0 hr, 4 hr, 8 hr, 24 hr.

Retrieved from "http://2012.igem.org/Team:Calgary/Notebook/Protocols/escapersassay"