wetbench author created giovannellilab

E.COLI CHEMICALLY COMPETENT CELLS (CaCl2)

Duration: 2h Adapted from: Sambrook & Russell,2006

Materials

  • Plate 90mm
  • Falcon tube 15mL
  • Flask 100mL
  • Eppendorf tube 1.5mL
  • Cuvette UV/VIS

Equipment

  • Dry incubator at 37°
  • Shaking incubator at 37°C
  • Spectrophotometer UV/VIS
  • Centrifuge

Solutions

  • CaCl2 50mM cold. sterilized
  • Glycerol 100%. sterilized
  • TY medium

Procedure

  1. Streak on agar plate (12h at 37°C-dry incubator)
  2. Inoculate a single colony in 5mL of TY liquid medium (~12h at 37°C-shaking incubator at 37°C)
  3. Inoculate 100mL of TY with the overnight culture for starting from OD600=0.1
  4. Incubate at 37°C until OD600=0.4-0.6 (approx. 2h)
  5. Transfer in 2 falcon 50mL of the culture and place on ice for 30 min
  6. Centrifuge at 3.500 rpm for 10' at 4°C. disardthge supernatant.
  7. GENTLY resuspend the pellet in 50mL ofl CaCl2
  8. entrifuge at 3.500 rpm for 10' at 4°C. dischard thge rnatant
  9. GENTLY resuspend the pellet in 25m CaCl2. Mix the 2 cultures into a single 50mL falcon.
  10. ncubate on ice for 15'.
  11. centrifuge at 3.500 rpm for 10' at 4°C. dishare the supesurnatant.
  12. resuspend the pellet GENTLY in 8.5mL of CaCl2 + 1.5mL Glycerol (1/10 of the initial volume).
  13. incubate 60' on ice.
  14. aliquot 400μL into sterile 1,5 mL tubes.
  15. tore at -80°C.

Expected results (quantitative information, graphics, images)

To test competent cell efficiency: transform an aliquote of competent cell with a plasmid carriyng a gene for antibiotic resistance

Common problems, troubleshooting and solutions

  • Low efficiency transformation which can be solved by changing molarity of CaCl2, using fresh CaCl2 or resuspending MORE gently