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>

Revision as of 17:32, 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
  • pour 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