Team:Calgary/Notebook/Protocols/potentiostatic

From 2012.igem.org

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<p>Similar to when creating a standard curve potentiostatically the working electrode must be held at a specific oxidation potential for the detection of that analyte. Testing for this requires a culture of cells that will be able to report through a hydrolase enzyme. To perform the detection a three electrode setup will be needed.</p>
<p>Similar to when creating a standard curve potentiostatically the working electrode must be held at a specific oxidation potential for the detection of that analyte. Testing for this requires a culture of cells that will be able to report through a hydrolase enzyme. To perform the detection a three electrode setup will be needed.</p>
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<ul><li>Grow cells overnight in 3mL of LB</li>
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<ol>
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<li>Pellet cells at 3750rpm for 10 minutes</li>
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<li>Grow cells overnight in 3mL of LB.</li>
-
<li>Remove supernatant</li>
+
<li>Pellet cells at 3750rpm for 10 minutes.</li>
-
<li>Resuspend in 1mL of 0.1M pH7 PBS</li>
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<li>Remove the supernatant.</li>
-
<li>Add to 25mL 0.1M pH7 PBS in an electrochemical cell</li>
+
<li>Resuspend in 1mL of 0.1M pH7 PBS.</li>
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<li>Add the solution to be tested for activation of the reporter gene</li>
+
<li>Add to 25mL 0.1M pH7 PBS in an electrochemical cell.</li>
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<li>Add the electrodes and close the cell</li>
+
<li>Add the solution to be tested for activation of the reporter gene.</li>
-
<li>Insert needle and bubble nitrogen or argon gas into the solution for 5 minutes</li>
+
<li>Add the electrodes and close the cell.</li>
-
<li>Hold the working electrode at the oxidation potential for the analyte product</li>
+
<li>Insert a needle and bubble nitrogen or argon gas into the solution for 5 minutes.</li>
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<li>Wait 5 minutes for current stabilization</li>
+
<li>Hold the working electrode at the oxidation potential for the analyte product.</li>
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<li>Add the sugar-analyte substrate</li>
+
<li>Wait 5 minutes for current stabilization.</li>
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<li>Observe any current changes over a time period</li>
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<li>Add the sugar-analyte substrate.</li>
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<li>Relate to concentration of analyte produced using a standard curve</li>
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<li>Observe any current changes over a time period.</li>
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</ul>
+
<li>Relate to the concentration of the analyte produced using a standard curve.</li>
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</ol>

Revision as of 22:31, 3 October 2012

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Reporter Expression Detection

Similar to when creating a standard curve potentiostatically the working electrode must be held at a specific oxidation potential for the detection of that analyte. Testing for this requires a culture of cells that will be able to report through a hydrolase enzyme. To perform the detection a three electrode setup will be needed.

  1. Grow cells overnight in 3mL of LB.
  2. Pellet cells at 3750rpm for 10 minutes.
  3. Remove the supernatant.
  4. Resuspend in 1mL of 0.1M pH7 PBS.
  5. Add to 25mL 0.1M pH7 PBS in an electrochemical cell.
  6. Add the solution to be tested for activation of the reporter gene.
  7. Add the electrodes and close the cell.
  8. Insert a needle and bubble nitrogen or argon gas into the solution for 5 minutes.
  9. Hold the working electrode at the oxidation potential for the analyte product.
  10. Wait 5 minutes for current stabilization.
  11. Add the sugar-analyte substrate.
  12. Observe any current changes over a time period.
  13. Relate to the concentration of the analyte produced using a standard curve.

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