Difference between revisions of "Team:Paris Bettencourt/Protocols/Electrocompetent Cells Preparation"

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     <td><b>Electrocompetent Cells Preparation Protocol</b>
 
     <td><b>Electrocompetent Cells Preparation Protocol</b>
 
     <ul>
 
     <ul>
 
       <li>Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
 
       <li>Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
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       <li>incubate until the the DO600 reach 0.5 to 0.7
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       <li>Incubate until the the DO600 reach 0.5 to 0.7
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       <li>place the cultures on ice for 15 minutes
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       <li>Place the cultures on ice for 15 minutes
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       <li>pour the culture in cold sterile 50mL falcon tubes
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       <li>For the culture in cold sterile 50mL falcon tubes
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       <li>centrifuge them for 10 minutes at 6000rpm
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       <li>Centrifuge them for 10 minutes at 6000rpm
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       <li>throw the supernatant
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       <li>Throw the supernatant
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       <li>resuspend the cells in 50mL cold distilled water
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       <li>Resuspend the cells in 50mL cold distilled water
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       <li>centrifuge them for 10 minutes at 6000rpm
+
       <li>Centrifuge them for 10 minutes at 6000rpm
−
       <li>throw the supernatant
+
       <li>Throw the supernatant
−
       <li>resuspend the cells in 25mL cold distilled water
+
       <li>Resuspend the cells in 25mL cold distilled water
−
       <li>centrifuge them for 10 minutes at 6000rpm
+
       <li>Centrifuge them for 10 minutes at 6000rpm
−
       <li>throw the supernatant
+
       <li>Throw the supernatant
−
       <li>resuspend the cells in 12.5mL cold 10% glycerol
+
       <li>Resuspend the cells in 12.5mL cold 10% glycerol
−
       <li>centrifuge them for 10 minutes at 6000rpm
+
       <li>Centrifuge them for 10 minutes at 6000rpm
−
       <li>throw the supernatant
+
       <li>Throw the supernatant
−
       <li>resuspend the cells in 5mL cold 10% glycerol
+
       <li>Resuspend the cells in 5mL cold 10% glycerol
−
       <li>make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C
+
       <li>Make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C
 
     </ul>
 
     </ul>
 
     </td>
 
     </td>
 
   </tr>
 
   </tr>
 
</table>
 
</table>

Latest revision as of 17:33, 14 August 2015

Electrocompetent Cells Preparation Protocol
  • Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
  • Incubate until the the DO600 reach 0.5 to 0.7
  • Place the cultures on ice for 15 minutes
  • For the culture in cold sterile 50mL falcon tubes
  • Centrifuge them for 10 minutes at 6000rpm
  • Throw the supernatant
  • Resuspend the cells in 50mL cold distilled water
  • Centrifuge them for 10 minutes at 6000rpm
  • Throw the supernatant
  • Resuspend the cells in 25mL cold distilled water
  • Centrifuge them for 10 minutes at 6000rpm
  • Throw the supernatant
  • Resuspend the cells in 12.5mL cold 10% glycerol
  • Centrifuge them for 10 minutes at 6000rpm
  • Throw the supernatant
  • Resuspend the cells in 5mL cold 10% glycerol
  • Make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C