Team:SDU-Denmark/Protocols/Competent-cells

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(Difference between revisions)
(→Protocol for making cells (E. coli) competent)
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=Protocol for making cells (E. coli) competent=
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=Protocol for making competent cells (E.coli)=
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(Cells are kept on ice at all times!! If the temperature rises above ~5˚C they lose their competency)
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#     2ml over-night cell culture is transferred to 200ml Luria Bertani (LB)-medium.
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  # 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium.
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#     Incubate at 37˚C on shaker until OD550=0.5-0.7
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  # Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
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#     Keep on ice for 15-30min and harvest by 4000 x g for 15 min at 4˚C.
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  # Cool cells on ice.
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#     Remove all supernatant and resuspend carefully in 200ml ice cold dH2O. Harvest as in 3.
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  # Harvest the cells in screwcap tubes (4 × 10 ml).
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#     Resuspend in 100ml ice cold dH2O. Harvest as in 3.
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  # Pour away the supernatant and keep the pellet on ice.
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#     Resuspend in 20ml ice cold 10% glycerol. Harvest as in 3.
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  # Wash the cells with 10 ml cold 50mM CaCl2.
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#     Resuspend in ice cold 10% glycerol until a total volume of 1ml. Cell concentration should be 1-3x1010 cells/ml.
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  # The cells are being distributed in eppendorf tubes of 200 µl.
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#     Cells are distributed in tubes with 40ul in each and kept at -80˚C.
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  # Add 41.7 µl 87% glycerol and mix well.
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  # Store at -80 º C.

Revision as of 15:18, 24 July 2009

Protocol for making competent cells (E.coli)

  # 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium.
  # Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
  # Cool cells on ice.
  # Harvest the cells in screwcap tubes (4 × 10 ml).
  # Pour away the supernatant and keep the pellet on ice.
  # Wash the cells with 10 ml cold 50mM CaCl2.
  # The cells are being distributed in eppendorf tubes of 200 µl.
  # Add 41.7 µl 87% glycerol and mix well.
  # Store at -80 º C.
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