DNA Sequencing of Plasmids

In double-stranded DNA sequencing, the two DNA strands must first be separated to enable the primer to bind to the priming site. This may be done by treating the DNA with alkali. Conventionally, use of the alkali denaturation method involves neutralization of the sample by acid treatment followed by ethanol precipitation and recovery of the DNA by centrifugation. The time-consuming process of ethanol precipitation may be avoided by neutralizing the alkali-treated DNA and recovering the sample in its original volume by passage through a spin-dialysis column. The use of spin-dialysis confers the additional advantage of cleaning up the template by removing traces of low-molecular-weight compounds that may interfere with the sequencing reactions. The method used here to prepare plasmid is a variation on the boiled-lysis method (1 ), using a rapid approach to avoid phenol extraction.