A Reliable,Efficient Protocol for 96-Well Plasmid DNA Miniprep with Rapid DNA Quantification for High-Throughput Automated DNA Sequencing

ABSTRACT

A procedure using a 96-well format for growth, template preparation, and quantification was optimized to facilitate high-throughput automated sequencing of double stranded plasmid DNA templates. Modification of the 96-well Miniprep Kit [Advanced Genetic Technologies (AGTC), Gaithersburg, MD] protocol combined with the DNA quantification with a Millipore Cytofluor 2350 (Millipore, Bedford, MA) provided high-quality DNA plasmid templates rapidly, efficiently, and inexpensively. We utilized this procedure to prepare more than 130,000 templates for a number of large-scale cDNA and genomic projects. Four specific projects were: human expressed sequence tag (EST) project (Adams et al., 1994) (70,080 templates), Haemophilus influenzae (Fleischmann et al., 1995) (22,656 templates), Mycoplasma genitalium (Fraser et al., 1995) (5760 templates), and Methanococcus jannaschii (22,175 templates). Data from these projects support a sequencing success rate of 89%–95%, with an average read length of 432 bases with <1% ambiguous bases.