Pham T T, Chillapagari S, Suarez A R
CLONTECH Laboratories, Inc., Palo Alto, CA, USA.
Biotechniques. 1996 Mar;20(3):492-7. doi: 10.2144/19962003492.
A rapid method has been developed for ultrapure plasmid or cosmid DNA isolation from ten-mL to several hundred-mL cultures of Escherichia coli (midi to maxi prep). A cleared lysate is prepared by alkaline lysis, followed by a quick alcohol precipitation step. Denatured bacterial DNA and RNA having at least 20 nucleotides of single-stranded regions are removed from the supercoiled plasmid by binding strongly to the single-strand affinity matrix (SSAMTM). Plasmid DNA is then effectively purified on a gel-filtration spin column to remove SSAM, proteins, small RNA and salts. This method produces consistent yields of high-quality plasmids that are suitable for use in many molecular biology applications. In addition, recombinant cosmids of approximately 46 kb can be purified intact, free of chromosomal DNA.
已开发出一种快速方法，用于从10毫升至数百毫升的大肠杆菌培养物中分离超纯质粒或黏粒DNA（中量至大量制备）。通过碱裂解制备清亮裂解物，随后进行快速乙醇沉淀步骤。通过与单链亲和基质（SSAMTM）强烈结合，将具有至少20个核苷酸单链区域的变性细菌DNA和RNA从超螺旋质粒中去除。然后在凝胶过滤离心柱上有效纯化质粒DNA，以去除SSAM、蛋白质、小RNA和盐。该方法能产生一致产量的高质量质粒，适用于许多分子生物学应用。此外，约46 kb的重组黏粒可完整纯化，不含染色体DNA。
