Application of silica-magnetite nanocomposites to the isolation of ultrapure plasmid DNA from bacterial cells

The aim of this study was to develop a simple and rapid method for purification of ultrapure plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe3O4) were prepared by chemical precipitation method using Fe2+, Fe3+ salt, and ammonium hydroxide under a nitrogen atmosphere. Silica-magnetite nanocomposites were prepared by the method of acid hydrolysis of tetraethoxysilane (TEOS) to coat the silica onto magnetite nanoparticles. DNA was adsorbed to the support under high salt conditions, and recovered directly in water for immediate downstream application, without the need for precipitation. We demonstrated that a useful plasmid, pRSETB-EGFP, encoding for the green fluorescent protein with T7 promoter, could be amplified in Escherichia coli of DE3 strain. Up to approximately 43 mu g of high-purity (A(260)/A(280) ratio = 1.75) plasmid DNA was isolated from 3 ml of an overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and polymerase chain reaction (PCR) amplification with success. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmid takes less than 8 min. The silica-magnetite nanocomposites deliver significant time-savings, overall higher yields, lower RNA contamination, and better PCR amplification compared to commercial available silica-based and other methods. (C) 2006 Elsevier B.V. All rights reserved.