Colman A, Byers M J, Primrose S B, Lyons A
Eur J Biochem. 1978 Nov 2;91(1):303-10. doi: 10.1111/j.1432-1033.1978.tb20966.x.
A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.
本文描述了一种快速制备分子量高达14×10⁶的质粒DNA的方法。该方法包括在室温下，在高浓度磷酸盐和尿素存在的条件下，将细菌裂解液上清在羟基磷灰石上进行层析。通过此步骤可去除质粒DNA中所有可检测到的蛋白质和RNA污染物，且质粒DNA的构象不受影响。纯化产物中染色体DNA的含量低于0.5%，如有必要，通过简单扩展该步骤以纳入热变性步骤，甚至可以去除这部分染色体DNA。该方法极其快速，适用于大规模质粒制备；40分钟内已纯化出5mg ColE1 DNA。质粒DNA的产量与传统染料离心技术相当，但纯度更高。
