High-throughput plasmid DNA purification for 3 cents per sample

To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical diffi- culty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week. 1 ml aliquots of bacterial cultures grown to saturation in 50 ml culture volumes. Bacterial cell pellets, collected in individual microfuge tubes, were resuspended in a lysozyme-containing solution. Exposure to microwave radiation was performed, and the resulting DNA purified by isopropanol precipitation. The authors noted that DNA prepared by their protocol was of quality suitable for restriction digests and other unspecified enzymatic modifications, but the suitability of the DNA for sequencing was not reported. Another study (3) reported the use of microwave radiation in the isolation of plasmid DNA from large volumes (250 ml) of bacterial cell cultures. Again, the suitability of this DNA for sequencing was not discussed. Both the volumes and formats reported in these studies were unsuited to large-scale sequencing; hence, we sought to adapt microwave-mediated bacterial cell lysis to 96-well format, and assess the performance of the resulting DNAs in sequencing reactions. Culturing plasmid clones and DNA purification