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Isolation of high-quality genomic DNA is a crucial step in bacterial comparative genomic studies using microarrays. Numerous procedures employing either physical or detergent/ enzyme-based cell lysing followed by phenol extraction, chaotropic-based fractionation, or size exclusion column purification can be used (1–4). However, few of them can be easily adapted for high-throughput operation at low cost. Despite the use of multiple time-consuming steps, DNA preparation remains a manageable task, since only a limited number of strains are used in most microarray experiments. We recently developed a new application of microarray technology, Library on a Slide, for bacterial comparative genomics studies (5). This platform combines the approach of dot blot hybridization with the technology of microarrays, resulting in glass slides arrayed with thousands of bacterial genomes (up to 15,000). Thus, libraries of entire genomes rather than the sequence of a single genome or set of genes are printed on the slides, which are used to screen these libraries for the presence of specific genetic elements of interest. An effective high-throughput procedure for DNA isolation from numerous samples is necessary for array printing. We previously used an UltraClean-htp™ 96-well microbial DNA isolation kit (Mobio, Carlsbad, CA, USA) to isolate DNA. While good-quality DNA was obtained, this time-consuming multistep protocol with relative low yield and high cost made it difficult to implement in preparing thousands of samples. This paper describes a new and simple method for preparing highly concentrated and fragmented bacterial DNA based on sonication and heat treatment. We have applied this new method with good success to a number of Gram negative and positive bacteria, including Escherichia coli, Hemophilus influenzae, Salmonella typhimurium, Klebsiella pneumoniae, Streptococcus agalactiae, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus mutans, and Enterococcus faecalis. Bacterial strains were grown overnight in 3–8 mL of the liquid medium of choice in 10-mL culture tubes for small batch processing or in 96-deep-well plates (two plates with 1.5 mL/well inoculants were later combined) for high-throughput processing. Bacteria were pelleted by centrifugation at 2000× g for 20 min and resuspended in 80 μL sonication buffer (50 mM Tris and 10 mM EDTA, pH 7.5, with optional 100 ng/μL RNase A). The suspension was transferred to a 0.5-mL thin wall PCR tube or a fully skirted 96-well PCR plate (Greiner Bio-One, Longwood, FL, USA). Then the tube/plate was placed in a plate horn (Misonix, Farmingdale, NY, USA) filled with a water and ice mixture and treated with …

Bacterial genomic DNA isolation using sonication for microarray analysis