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MICROBIOLOGY MIMM 386 Laboratory Course in Microbiology and Immunology

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

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Title: MICROBIOLOGY MIMM 386 Laboratory Course in Microbiology and Immunology


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

2
(No Transcript)
3
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

4
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

5
Circular vs supercoiled plasmids
6
Transformation efficiency in different conditions
7
DH5? cells
Inoculate from a single colony
Grow at 37C 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
8
E. coli DH5? growth curve in LB broth
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