A simplified and improved method for the efficient double-stranded sequencing of mini-prep plasmid DNA. Nucleic Acids Res

Mini-prep of plasmid DNA followed by restriction mapping and sequencing is useful for analyzing a DNA fragment of interest. Numerous methods for fast mini-prep of DNA have been proposed, but the isolated DNA is either not pure enough for sequencing (1, 4, 6), or the procedures involved the use of enzyme inhibiting denaturating agents (2, 7), enzymes treatments (3), or multi-step DNA precipitation (5), which are time consuming and tedious. A 20 min DNA mini-prep kit is commercially available (LKB-Pharmacia), however, fine adjustment of the kit to suit different purposes is not possible. By combining alkaline lysis and salt precipitation of high MW RNA into one step, removal of low MW RNA and protein into the second step, mini-prep of plasmid DNA suitable for sequencing can be done within 20 min. Centrifuge 1.5 ml saturated O/N culture at 12000 g for 1 min to collect the bacteria. Resuspend the pellet in 100 /d buffe