To improve the efficiency of transformation, E. coli cells are suspended in cold calcium chloride, then subjected to 42oC heat shock for a short period. Cells in the early- to mid-log stage of growth respond best to this treatment, having efficiencies ranging from 105 to 107 transformants per microgram (µg) of plasmid DNA (~1/100,000 of all viable cells.).
Other ions such as cobalt (Co++), manganese (Mn++), magnesium (Mg++), and barium (Ba++) can also increase transformation efficiency, and when used as a mixture along with a reducing agent as a buffer can achieve up to 109 transformants per µg of plasmid (~1/10th of all viable cells).
Since transformation is limited by the number of competent cells, increasing the concentration of plasmid does not increase efficiency. Raising plasmid concentration higher than about 0.1 to 0.2 µg per ml of mid-log cells may actually decrease efficiency, though competent cells can absorb more than one plasmid.