Team:SDU-Denmark/Protocols/Competent-cells

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Protocol for making competent cells (E.coli)

(Cells are kept on ice at all times!! If the cells temperature rises above ~5º C they'll lose their competency!)

  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 -80º C.