Team:Tuebingen/Notebook/Protocols/tecanreader

From 2013.igem.org

(Difference between revisions)
Line 17: Line 17:
<div id="actualContent">  
<div id="actualContent">  
-
<p>The Tecan reader is used for flourescence spectroscopy. Thus, tested cells should express some sort of flourescent protein. We mainly tested yeast cells that express mOrange - <a href="http://parts.igem.org/Part:BBa_E2050">BBa_E2050</a>.</p>
+
<p>The Tecan reader is used for flourescence spectroscopy. Thus, tested cells should express some sort of flourescent protein. We mainly tested yeast cells that express mOrange (<a href="http://parts.igem.org/Part:BBa_E2050">BBa_E2050</a>).</p>
<h3>Procedure</h3>
<h3>Procedure</h3>
<ol>
<ol>
-
   <li>Inoculate 5 mL YPD medium with yeast cells. Create 1:10 and 1:100 dilutions of this liquid culture. Store all at 30°C.</li>
+
   <li>Inoculate 5 mL liquid YPD medium with yeast cells. Create 1:10 and 1:100 dilutions of this liquid culture. Incubate all cultures at 30°C over night.</li>
-
   <li></li>
+
   <li>On the next day, check OD600 (culture density). Ideally, OD600 is between 1 - 1.5 (maximum: 2.0).</li>
-
   <li></li>
+
   <li>Transfer cells to Eppendorf tubes and centrifuge at 13 000 rpm for 30 sec.</li>
-
   <li></li>
+
   <li>Discard supernatant and add 1 mL Aqua dest. Resuspend cells.</li>
-
   <li></li>
+
   <li>Inoculate 1/3 of initial culture volume (minimum: 600 µL) liquid YPD (OD600 should be between 3 - 5) with cells from previous step.</li>
-
   <li></li>
+
   <li>Transfer 150 µL cell suspension in a black 96-well-plate. Create a 150 µL water blank.</li>
-
   <li></li>
+
   <li>Measure cells in Tecan-Reader. Adjust settings according to your requirements. We have used:
 +
    <ul>
 +
        <li>Plate: Greiner 96 Flat Bottom Black</li>
 +
        <li>Mode: Flourescence intensity</li>
 +
        <li>Excitation: 548 nm</li>
 +
        <li>Emission: 581 nm</li>
 +
        <li>Gain: 150</li>
 +
        <li>Number of flashes: 25</li>
 +
        <li>Time of integration: 25 µs</li>
 +
    </ul>
 +
  </li>
   <li></li>
   <li></li>
</ol>
</ol>

Revision as of 20:07, 4 October 2013

Return to iGEM Main Page.

Tecan Reader

The Tecan reader is used for flourescence spectroscopy. Thus, tested cells should express some sort of flourescent protein. We mainly tested yeast cells that express mOrange (BBa_E2050).

Procedure

  1. Inoculate 5 mL liquid YPD medium with yeast cells. Create 1:10 and 1:100 dilutions of this liquid culture. Incubate all cultures at 30°C over night.
  2. On the next day, check OD600 (culture density). Ideally, OD600 is between 1 - 1.5 (maximum: 2.0).
  3. Transfer cells to Eppendorf tubes and centrifuge at 13 000 rpm for 30 sec.
  4. Discard supernatant and add 1 mL Aqua dest. Resuspend cells.
  5. Inoculate 1/3 of initial culture volume (minimum: 600 µL) liquid YPD (OD600 should be between 3 - 5) with cells from previous step.
  6. Transfer 150 µL cell suspension in a black 96-well-plate. Create a 150 µL water blank.
  7. Measure cells in Tecan-Reader. Adjust settings according to your requirements. We have used:
    • Plate: Greiner 96 Flat Bottom Black
    • Mode: Flourescence intensity
    • Excitation: 548 nm
    • Emission: 581 nm
    • Gain: 150
    • Number of flashes: 25
    • Time of integration: 25 µs

 

Back to Protocols


Retrieved from "http://2013.igem.org/Team:Tuebingen/Notebook/Protocols/tecanreader"