User:Daniel Goodman/Notebook/Cluzel/2010/03/31

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Protocol for Competent Cells
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*Grow 250 ml flask, one colony, 50 ml LB at 37 deg for 4 hours (OD must be 0.3-0.4) in shaker (check at 7pm)
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*Spin down 20 mls in 2 50 ml epis at 4 degrees in centrifuge
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**3200 RCF (4000 RPM) 10 minutes 4 degrees
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** pour of supernatant, add 40 mls 4 deg filtered water each (maybe add 5 first, vortex, add 35 more)
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*** make sure pellet is dissolved
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** repeat above 2x
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* resuspend pellets in 10% glycerol (5 mls each)
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* spin, remove glycerol supernatant
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* resuspend in 0.5 ml 10% glycerol each, combine tubes
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* make 20x 50 ul aliquots, put in -80 freezer
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Revision as of 15:10, 31 March 2010


Cluzel Lab Notebook
Daniel Goodman
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Entry title

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Protocol for Competent Cells

  • Grow 250 ml flask, one colony, 50 ml LB at 37 deg for 4 hours (OD must be 0.3-0.4) in shaker (check at 7pm)
  • Spin down 20 mls in 2 50 ml epis at 4 degrees in centrifuge
    • 3200 RCF (4000 RPM) 10 minutes 4 degrees
    • pour of supernatant, add 40 mls 4 deg filtered water each (maybe add 5 first, vortex, add 35 more)
      • make sure pellet is dissolved
    • repeat above 2x
  • resuspend pellets in 10% glycerol (5 mls each)
  • spin, remove glycerol supernatant
  • resuspend in 0.5 ml 10% glycerol each, combine tubes
  • make 20x 50 ul aliquots, put in -80 freezer


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