Preparation of competent cells and transformation of E.coli
Nucleic acids do not enter bacteria under their own power, but require assistance traversing the outer and inner cell membranes and in reaching an intracellular site where they can be expressed and replicated. Chemical methods can be used to make the cells competent and transform them.
E.coli cells washed in cocktail of simple salt solutions achieve a state of competence during which DNA molecules may be admitted to the cell. Bacteria treated with ice cold solutions of CaCl2 and then briefly heated to 37ºC or 42ºC could be transferred with DNA.
Transformation of different bacterial strains by plasmid DNA involves the use of complex cocktails of divalent cations in different buffers, treating cells with reducing agents, adjusting the ingredients of the cocktail to the genetic constitution of particular strains of E.coli, harvesting cells at specific stages of growth cycle, altering the temperature of growth of culture before exposure to chemicals, optimizing the extent and temperature of heat shock, freezing and thawing cells and exposing cells to organic solvents after washing in divalent cations. By all these treatments transformation frequencies ranging from10 (6) to 10(9) transformants/µg of super helical plasmid DNA can be achieved.