用SephacrylS-1000柱层析大量纯化质粒DNA

PREPARATION OF HIGH PURITY PLASMID DNA BY USING SAPHACRYLS-1000 GEL COLUMNS CHROMATOGRAPHY

作者介绍一种用Sephacryls-1000凝胶柱层析纯化质粒DNA的简便、有效方法。该实验程序中既不用RNA酶处理,也不需氯化铯-溴乙锭(CsCl-EB)密度梯度离心。回收的质粒DNA中无RNA和染色体DNA污染,绝大部分是共价闭环DNA(ccc DNA),只含少量开环DNA(oc DNA),没有线型DNA。并保持其正常的生物活性和分子结构。

We report here a simple, rapid and efficient procedure for large-scale purificiation of plasmid DNA. It is a modification of the alkaline lysis method of Maniatis T, et al. Cell lysates are deproteinized, precipitaed with CaCl_2 to remove rRNA, concentrated by ethanol precipitation, and applied to gelfiltratration chromatography by sephacryl S-1000 column for final purification of plasmid DNA. The modified method does not require CsClEB densitygradient centrifugation; the recovered plasmids are free of RNA and chromosomal DNA; cccDNA are greater than 80%and retain their normal biological activity and molecular component. The yields of 600-800μg of high purity plasmid DNA are obtained from Ⅰ liter amplified culture. SephacrylS-1000 gel columns can be reused repeatedly after washing with 1-2 column volume elution buffer. The method is also inexpensive.