Difference between revisions of "Team:Bielefeld-CeBiTec/Protocols"

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                     <p>...</p>
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<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>
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</ul>
 
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Revision as of 15:15, 12 August 2015

iGEM Bielefeld 2015


Protocols



PCR

...

  • 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