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MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology)

MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology). Exercise 4. Dr. Benoit Cousineau Department of Microbiology & Immunology McGill University. Exercise 4 : Preparation of competent cells and transformation of E. coli with plasmid DNA. Transformation of bacteria:

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MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology)

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  1. MICROBIOLOGY MIMM 386(Laboratory Course in Microbiology and Immunology) Exercise 4 Dr. Benoit Cousineau Department of Microbiology & Immunology McGill University

  2. Exercise 4: Preparation of competent cells and transformation of E. coli with plasmid DNA • Transformation of bacteria: • Introduce a plasmid within the cell (cytoplasm) • Competent cells: • Cells treated to increase their efficiency for DNA uptake from the medium • Natural competence is too low • The mechanism is still unknown (empirical data) • Transient state of competence

  3. Different strategies to transform bacterial cells • Heat shock: • Ice-cold solution of CaCl2 (2-mercaptoethanol) • 42ºC for 45 seconds (cells + plasmid DNA) • 105-106 colonies/µg of supercoiled plasmid DNA • Plasmid size and form can affect the efficiency • Electroporation: • Ice-cold water • 2.5kVolts at 4ºC (cells + plasmid DNA) • 109-1010 colonies/µg of supercoiled plasmid DNA • Plasmid size and form can affect the efficiency

  4. Circular vs supercoiled plasmids

  5. Transformation efficiency in different conditions

  6. DH5 cells Inoculate from a single colony Grow at 37°C OD600 = 0.45-0.55  10µl or less 42ºC for 45 seconds 2.5 kVolts at 4ºC Add 900 µl of SOC, incubate 1h00 (37ºC), dilute and spread on LB/Amp plates

  7. E. coli DH5 growth curve in LB broth

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