Team:Tuebingen/Notebook/Protocols/competent cells

(Difference between revisions)

Revision as of 20:52, 14 October 2014

It is generally to be considered to keep the bacteria at 0 °C. The equipment has to be cooled on ice before usage.

2.0 ml of a bacterial pre-culture are transferred to 100 ml of 37 °C warm LB medium
Incubate at 37 °C until the culture reaches a OD600/ml of ca. 0.5
Transfer the culture with the required OD in 50 ml culture tubes
Centrifuge for 5 min with 5000 rpm at 4 °C
Resuspend the pellet in 15 ml cold and sterile 100 mM CaCl2 and incubate for 30 min on ice
Centrifuge for 5 min with 5000 rpm at 4 °C
Resuspend the pellet in 4 ml cold and sterile 1 M CaCl2 and incubate for 15 min on ice
Add 840 μl 87 % (w/v) glycerine (end concentration of 15 %)
Aliquot 200 μl of the cell suspension in Eppendorf-tubes and freeze in liquid nitrogen. Store at -80 °C.

@@ Line 12: / Line 12: @@
 <h3>Creation of competent <i>Escherichia coli</i> cells</h3>
-<p> </p>
+<p>It is generally to be considered to keep the bacteria at 0 °C. The equipment has to be cooled on ice before usage.</p>
+<ol>
+  <li>2.0 ml of a bacterial pre-culture are transferred to 100 ml of 37 °C warm LB medium</li>
+  <li>Incubate at 37 °C until the culture reaches a OD600/ml of ca. 0.5</li>
+  <li>Transfer the culture with the required OD in 50 ml culture tubes </li>
+  <li>Centrifuge for 5 min with 5000 rpm at 4 °C</li>
+  <li>Resuspend the pellet in 15 ml cold and sterile 100 mM CaCl2 and incubate for 30 min on ice</li>
+  <li>Centrifuge for 5 min with 5000 rpm at 4 °C</li>
+  <li>Resuspend the pellet in 4 ml cold and sterile 1 M CaCl2 and incubate for 15 min on ice</li>
+  <li>Add 840 μl 87 % (w/v) glycerine (end concentration of 15 %)</li>
+  <li>Aliquot 200 μl of the cell suspension in Eppendorf-tubes and freeze in liquid nitrogen. Store at -80 °C.</li>
 </div>