Factors which influence CaC12 dependent transfection of lambda DNA in Escherichia coli K12 recipients

This paper presents further parameters influencing the competence, the process of DNA uptake and the efficiency of plating of CaC12-treated E. coli D12 strains. We have found that the process of DNA uptake depends not only on the treatment of bacteria with a certain CaCl2-concentration but is also influenced considerably by a shift-down of the CaCl2-concentration in the reaction mixture. The pH of the growth media and of the reaction mixture plays an important role in maintaining of optimal transfection. The efficiency of plating is influenced by the thickness of the top layer and the concentration of bacteria on the plate. Without genetic variation of the strains, by only varying the mentioned factors we could improve the efficiency of CaCl2 transfection at about two orders of magnitude to a maximum of 6 X 105 pfu/microgram DNA.