¹ Clemson University Genomics Institute, Clemson, SC, 29634, USA, http://www.genome.clemson.edu

Recent advance in sequencing technologies has increased the overall throughput for large scale genomic sequencing project. This accelerated speed of genome DNA sequencing demands efficient, rapid and inexpensive methods. Although a number of methods for the plasmid DNA preparation they are not completely satisfactory in all the aspects; the high quality for sequencing, high throughput, low cost-performance, low cost-set up, low degree of technical difficulty, and automation In this report, we describe a more simple high-throughput plasmid DNA extraction procedure based on the 96-well filter plate and modified alkaline lysis method. The entire protocol can be completed in as little as 1 hour without requiring a cell harvest procedure. One technician with a 8 channel electric pipette and 2 centrifuges can process up to 1920 samples in 5 hours without automation. Pipetting, the most time consuming steps of this method, can be automated to significantly increase the throughput. This new method yields up to 2 micrograms of plasmid DNA from a 150¥ìl overnight culture in a 96-deep-well block. Initial results have shown templates prepared by this method to yield greater than 500 bp high quality bases with approximately 90% success ratio. This procedure is suitable for feeding the unprecedented scaling up of capillary sequencing, ideal for any high-throughput operation in terms of template quantity, quality, cost, and speed. It does not require expensive reagents or equipment, and amenable to automation because of the simplicity of the whole procedure. Together, the physical and transcript maps described here serve as basic resources for further genome study.