We have re-evaluated the conditions for preparing competent Escherichia coli cells and established a simple and efficient method (SEM) for plasmid transfection. Cells (DH5, JM109 and HB101) prepared by SEM are extremely competent for transformation (1-3 x 10(9) cfu/microgram of pBR322 DNA), and can be stored in liquid nitrogen for at least 40 days without loss of competence. Unlike electroporation, transformation using these competent cells is affected minimally by salts in DNA preparation. These competent cells are particularly useful for construction of high-complexity cDNA libraries with a minimum expenditure of mRNA.