Transformation of E.coli (according to Inouoe et al. (1990)

Reference:
Inoue et al. (1990). An efficient transformation of E. coli with plasmids. Gene 96, 23-28.

Protocol: Preparation of CC

  1. Streak E. coli cells on an LB agar plate
  2. Culture overnight at 37°C
  3. Isolate 10 to 12 large colonies (2-3 mm in diameter) with a loop
  4. Inoculate in 250 ml of SOB medium in a 2-l flask
  5. Grow to OD600 of 0,6 at 18°C (or room temperature 20°C) with shaking (200-250 rpm)
    ! This will take one day a half at 20°C !
  6. Place the flask on ice for 10 min
  7. Harvest the cells at 2500xg (3000 rpm) for 10 min at 4°C
  8. Resuspend the pellet in 80 ml of ice-cold TB
  9. Incubate on ice for 10 min
  10. Spin down as above
  11. Resuspent the pellet gently in 20 ml TB
  12. Add DMSO by gentle swirling to a final concentration of 7% (1,5 ml DMSO)
  13. Incubate on ice for 10 min
  14. Dispence by 500 µl into tubes
  15. freeze quickly in liquid nitrogen
  16. Store frozen competent cells at -70°C

Protocol: Tranformation of CC

  1. Transfer competent cells from -70°C on ice
    Let them thaw for 10 min
  2. Put 200 µl competent cells in a 15 ml Falcon tube (! not in a glass tube !)
  3. Take 1/10 volume of the ligation mix (up to 20 µl volume, not more than 10 ng DNA)
    mix with the competent cells
  4. Leave this on ice for 30 min,
    Prepare a water bath of 42°C and liquid SOC medium (with IPTG)
  5. Heat-shock the mixture without agitation for 30 sec at 42°C
  6. Tranfer immediately to an ice bath and leave it for 10 min
  7. Add 800 µl SOC and place the tubes at 37°C with vigorous shaking for 1 h
  8. Plate out 100 µl resp. 900 µl on selective LB plates
  9. Incubate plates at 37°C overnight

 

Media

SOB

SOC

LB

TB

10 mM Hepes (2,383 g/l)
 

55 mM MnCl2 (3,6 g/l)
 

15 mM CaCl2 (2,2 g/l)
 

250 mM KCl (18,6 g/l)
 

All components (solids), except MnCl2 are mixed and adjusted to pH 6,7 with KOH
MnCl2 is dissolved, sterilized by filtration (0,45 µm filter) and stored at 4°C (to prevent precipitation, in the mixture) (! carcinogenic !)

DMSO (! Prevent oxidation, aliquot the stock solution !)

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