The two DNA sequencing techniques currently in use are the chemical degradation method developed by Maxam and Gilbert (1979) and the enzymatic method developed by Sanger et al. (1977). Due to the less laborious protocol and the easy availability of oligonucleotides, the ‘Sanger’ method is now the most widely used one, being applicable not only for the sequencing of DNA fragments cloned in plasmid or M13 phage vectors but also, in combination with PCR, for the direct sequencing of minute amounts of template (Ellingboe and Gyllensten, 1992). This chapter describes a standard procedure for the sequencing of single- or double-stranded plasmid DNA, based on the ‘Sanger’ or ‘dideoxy’ method.