Techniques for transformation of <i>E. coli</i>

Abstract The transfer of plasmid DNA into E. coli, so-called ‘plasmid transformation’, is a cornerstone of modern molecular biology. The original observation that calcium ions induced a state of competence, whereby E. coli cells would take up DNA and establish it in the cell (1, 2), set the foundation for a number of techniques for inducing competence toward plasmid transformation. One class of techniques is founded on this original observation that Ca2+ and other divalent and multivalent cations can induce competence. More recently, an alternative technique, electroporation, involving application of a transient high voltage electrical field to a mixture of E. coli cells and plasmid DNA, has proved to be remarkably efficient. Electroshock transformation techniques currently produce the highest efficiencies of any known methodology. In this chapter we present a spectrum of techniques that together produce reliable transformations at differing efficiencies, and for distinctive purposes. These range from low efficiency colony transformations, which can be performed in a few minutes without advance preparation of competent cells, to electro shock transformations whereby high complexity plasmid libraries can be produced with minimal amounts of starting material. We also describe methods for properly storing E. coli strains that will be used for preparation of competent cells, as well as methods for producing frozen competent cells, and discuss other parameters which are important for reliable production of competent cells. This chapter is adapted from a more comprehensive presentation on methods for and genetic factors influencing plasmid transformation of E. coli and other bacteria (3).