Very rapid nucleotide sequence analysis of improved, double-stranded minipreps

Recently, the combination of double-stranded sequencing and Sequenase has been used to accomplish rapid, high-performance sequencing. This combination is relatively resistant to the usual compression effects of palindromes seen with sequencing enzymes such as Klenow fragment of DNA polymerase I or reverse transcriptase. However, for optimal results the method has still relied on plasmids purified by centrifugation through CsCl gradients. The preparation of large-scale cultures, CsCl gradients, and subsequent dialysis are time-consuming processes. The present report describes an improved, miniprep procedure which eliminates the need for CsCl gradients or single-stranded vectors. The effectiveness of the procedure is due to increased ampicillin concentration which amplifies the plasmids, destruction of contaminating enzymes by diethylpyrocarbonate treatment, and vortexing to facilitate rapid sample handling. Sequences of the resulting minipreps are equal in resolution and quality to sequences of CsCl-gradient-purified plasmids.