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Team:Tuebingen/Notebook/Protocols/competent cells
From 2014.igem.org
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| - | + | <h1>Protocols</h1> | |
<h3>Creation of competent <i>Escherichia coli</i> cells</h3> | <h3>Creation of competent <i>Escherichia coli</i> cells</h3> | ||
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| - | <li> 2.0 ml of a bacterial pre-culture are transferred to 100 ml of 37 °C warm LB medium</li> | + | <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 | + | <li> Incubate at 37 °C until the culture reaches a OD<sub>600</sub>/ml of ca. 0.5</li> |
| - | <li> Transfer the culture with the required OD in 50 ml culture tubes </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> 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> 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> Centrifuge for 5 min with 5000 rpm at 4 °C</li> |
| - | <li> Resuspend the pellet in 4 ml cold and sterile 1 M | + | <li> Resuspend the pellet in 4 ml cold and sterile 1 M CaCl<sub>2</sub> and incubate for 15 min on ice</li> |
| - | <li> Add 840 μl 87 % (w/v) glycerine (end concentration of 15 %)</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> | + | <li> Aliquot 200 μl of the cell suspension in Eppendorf-tubes and freeze in liquid nitrogen. Store at -80 °C.</li> |
</div> | </div> | ||
Latest revision as of 15:30, 17 October 2014
Protocols
Creation of competent Escherichia coli cells
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.
