Difference between revisions of "Team:Bielefeld-CeBiTec/Protocols"
Line 142: | Line 142: | ||
<div id="HeatShock" class="panel-collapse collapse"> | <div id="HeatShock" class="panel-collapse collapse"> | ||
<div class="panel-body"> | <div class="panel-body"> | ||
− | < | + | <ul> |
+ | <li>Thaw 100 µl chemo competent <i>E. coli</i> cells on ice </li> | ||
+ | <li>Add 0.5-5 µl plasmid to 100 µl chemocompetent cells </li> | ||
+ | <li>Store cells on ice for 10-30 min on ice </li> | ||
+ | <li>Heat shock for 90 seconds at 42 °C </li> | ||
+ | <li>Store reaction on ice for 60 seconds </li> | ||
+ | <li>Optional: Preheat SOC medium to 37 °C</li> | ||
+ | <li>Transfer reaction to 1 ml SOC medium and incubate at 37 °C for at least 1 hour</li> | ||
+ | <li>Centrifuge 3 minutes at 12000 rpm and plate on selective LB medium </li> | ||
+ | <li>Incubate at 37 °C over night </li> | ||
+ | |||
+ | </ul> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 15:15, 12 August 2015
Protocols
...
...
...
...
- Thaw 50 µl electrocompetent E. coli cells on ice, dilute with icecold 50 µl glycerine (10%) if necessary
- Add 0.5-5 µl plasmid to 50 µl electrocompetent cells
- Store cells on ice for 1 minute
- Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ω
- Transfer transformation reaction to 450 µl SOC-Medium and incubate 1 h at 37 °C
- Plate on selective LB-Medium
- Incubate over night at 37 °C
- Thaw 100 µl chemo competent E. coli cells on ice
- Add 0.5-5 µl plasmid to 100 µl chemocompetent cells
- Store cells on ice for 10-30 min on ice
- Heat shock for 90 seconds at 42 °C
- Store reaction on ice for 60 seconds
- Optional: Preheat SOC medium to 37 °C
- Transfer reaction to 1 ml SOC medium and incubate at 37 °C for at least 1 hour
- Centrifuge 3 minutes at 12000 rpm and plate on selective LB medium
- Incubate at 37 °C over night