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

3. Transformation of bacteria: Competent cells:
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)
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)
colonies/µg of supercoiled plasmid DNA

5. Circular vs supercoiled plasmids

6. Transformation efficiency in different conditions

7.  DH5 cells Inoculate from a single colony Grow at 37°C
OD600 = 
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