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