Paris/August 14


yesterday -- tomorrow

Transformation of a lox-KmR-lox strain with a pBAD-CRE plasmid

Francois Xavier Barre gave us the strain FX85 which as a lox-Km-lox in its chromosome. We will prepare CaCl2 competent cells of FX85 and transform it with a plasmid bearing pBAD-CRE. This will allow us measuring the excision frequency of the CRE/LOX system with different levels of induction

Competent Cells:

  • Grow bacteria from fresh streak in 30ml LB until OD 0.6
  • Cool by swirling in ice water. pour in 50ml tube and centrifuge 3000rpm for 10min at 4°C
  • Pour off supernatant and resuspend in 20ml ice-cold CaCl2 0.1M. Sit on ice 20min.
  • Centrifuge as before and resuspend in 0.5ml of Cacl2 0.1M. Sit on ice.


  • Mix 100µl of cells with 10µl DNA. Sit on ice 20min.
  • Heat shock 2min at 37°C. Put on ice 2min.
  • Add 2ml LB
  • Incubate 1H at 37°C
  • Plate 100µl and 1.5 ml

MiniPrep and glycerol stock

Plasmids: MiniPrep Products
Number Name Description Products in use Date
MP13 BBa_J61002 ptet-mRFP MP13.1, MP13.2 August 14
MP14 BBa_J61025 Medium constitutive promoter in J61002 (clones 1 + 2) MP14.1, MP14.2 August 14