Preparation of Single-Stranded DNA from Phagemid Vectors

Abstract

Single-stranded DNA (ssDNA) is the optimal template for most polymerase-based molecular-biology applications, including DNA sequencing and site-directed mutagenesis. Phagemids are chimeric vectors, derived from the ssDNA bacteriophages M13, fd, or f1, that normally replicate as plasmids in bacterial hosts (1) (see Chapter 2). Phagemids typically lack the genes required for packaging into virions and, thus, “helper” bacteriophage are often required to package phagemids as ssDNA into viral particles, which are subsequently released into the culture medium (2). ssDNA isolated from these virions is suitable for use as a template in a variety of procedures with no further manipulation, as denaturation is not required prior to its use as a template.