Team:SDU-Denmark/Protocols/Competent-cells

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(→Protocol for making competent cells (E.coli))
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=Protocol for making competent cells (E.coli)=
=Protocol for making competent cells (E.coli)=
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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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# Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
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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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# Cool cells on ice.
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  # Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
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# Harvest the cells in screwcap tubes (4 × 10 ml).
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  # Cool cells on ice.
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# Pour away the supernatant and keep the pellet on ice.
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  # Harvest the cells in screwcap tubes (4 × 10 ml).
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# Wash the cells with 10 ml cold 50mM CaCl2.
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  # Pour away the supernatant and keep the pellet on ice.
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# The cells are being distributed in eppendorf tubes of 200 µl.
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  # Wash the cells with 10 ml cold 50mM CaCl2.
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# Add 41.7 µl 87% glycerol and mix well.
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  # The cells are being distributed in eppendorf tubes of 200 µl.
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# Store at -80 º C.
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  # Add 41.7 µl 87% glycerol and mix well.
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  # Store at -80 º C.
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Revision as of 15:18, 24 July 2009

Protocol for making competent cells (E.coli)

  1. 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium.
  2. Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
  3. Cool cells on ice.
  4. Harvest the cells in screwcap tubes (4 × 10 ml).
  5. Pour away the supernatant and keep the pellet on ice.
  6. Wash the cells with 10 ml cold 50mM CaCl2.
  7. The cells are being distributed in eppendorf tubes of 200 µl.
  8. Add 41.7 µl 87% glycerol and mix well.
  9. Store at -80 º C.
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