大质粒DNA提纯方法的改进

本文报道了一种源于Kado-Liu法经系列改进后产生的大质粒DNA检测与提取方法。改进之处主要是:裂解液组成中的Tris度为500mM,pH值为12.8～13.0;蛋白质的抽提除了酚—氯仿外同时使用了适量的醋酸钠(3M pH4.8)溶液;以异丙醇室温静置30分钟左右,取代冷乙醇较长时间的低温处理,进行DNA浓缩。该方法在肠道菌方面的应用中,显示了其简易、快速和较好的重复性。另外,用930～950μl新鲜10N NaOH液使裂解液pH值为最佳的经验值,既可避免因不同类型pH计产生不同结果的麻烦,亦为该方法能在普通实验室推广应用提供了方便。

Improved Method for the Extraction of Large Plasmid DNA

A method for extraction of the large plasmid DNA was developed by improving of Kado-Liu method. The characteristics of this improved method are: Both Tris concentration and pH value of the solution for lysing are higher than that of the original method, 500 mM and 12.8～13.0 respectively; proper amount sodium acetate was used for extraction of proteins in addition to phenol-chloroform; isopropanol was used to concentrate the target DNA at room temperature instead of the cold ethanol. The improved methed showed that it was simple and rapid, and gave rise reproducible results in detection and extraction of plasmid DNA in enterobacterial species.The pH of solution used for lysing can be adjusted directly by adding 930～950μ1 of 10N NaOH (freshly prepared) in 100 ml Tris-SDS solution, which may enable this method more conveniently used in common laboratories.