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Simple methods for preparation of plasmid DNA yielding long and accurate sequence data.

DNA sequencing results largely depend on the quality of templates. Sequencing with fluorescent primers is especially sensitive to the quality of the DNA. Usual readable length is about 200-300 bp for plasmid DNA isolated either by the miniprep method of Serghini et al. (1) or by the alkaline lysis methods of Birnboim and Doly (2). To obtain sequence data up to 500 bp, we must use templates purified either by equilibrium centrifugation in CsCl-ethidium bromide gradients (3) or by binding to silica-based resins (4). We have found that addition of MgCl2 and/or chilling at -80°C improve the quality of the plasmid DNA preparation. Templates purified by either of the following methods provide reproducible fluorescent sequencing results comparable to those obtained by resin-purified plasmid DNA.