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

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

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

Circular vs supercoiled plasmids

Transformation efficiency in different conditions

 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

E. coli DH5 growth curve in LB broth