Reddy K J, Kuwabara T, Sherman L A
Division of Biological Sciences, University of Missouri, Columbia 65211.
Anal Biochem. 1988 Feb 1;168(2):324-31. doi: 10.1016/0003-2697(88)90325-9.
A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.
本文描述了一种简单快速的方法来纯化大量的λ噬菌体颗粒和DNA。该方法包括对噬菌体颗粒进行DEAE - 纤维素柱层析，并用低离子强度缓冲液从柱上洗脱噬菌体颗粒。所得噬菌体与源自大肠杆菌宿主细胞的RNA、DNA和蛋白质得到了很好的分离。λDNA是通过常规的酚抽提和乙醇沉淀方法从纯化的噬菌体颗粒中制备的。该方法不使用核酸酶、蛋白酶、去污剂或CsCl密度梯度离心。通过该方法获得的λDNA纯度与通过CsCl密度梯度离心制备的材料相当，并且适合进行限制性酶切、连接、放射性标记和双链DNA测序。文中详细描述了一个在不到5小时内从1升液体裂解物中获得0.5至1.0毫克DNA的方案。该方法简单、廉价且省时，特别适合大规模分离λDNA。
