Team:SDU-Denmark/Protocols/Competent-cells

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=Protocol for making cells (E.coli) competent for transformation=

(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.

=Protocol for making cells (E. coli) competent for electroporation=

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


 * 1)      2ml over-night cell culture is transferred to 200ml Luria Bertani (LB)-medium.
 * 2)      Incubate at 37˚C on shaker until OD450=0.5-0.7
 * 3)      Keep on ice for 15-30min and harvest by 4000 x g for 15 min at 4˚C.
 * 4)      Remove all supernatant and resuspend carefully in 200ml ice cold dH2O. Harvest as in 3.
 * 5)      Resuspend in 100ml ice cold dH2O. Harvest as in 3.
 * 6)      Resuspend in 20ml ice cold 10% glycerol. Harvest as in 3.
 * 7)      Resuspend in ice cold 10% glycerol until a total volume of 1ml. Cell concentration should be 1-3x1010 cells/ml.
 * 8)      Cells are distributed in tubes with 40ul in each and kept at -80˚C.