A SIMPLE AND EFFICIENT PROCEDURE FOR THE ISOLATION OF HIGH-QUALITY PHAGE-LAMBDA DNA USING A DEAE-CELLULOSE COLUMN
A SIMPLE AND EFFICIENT PROCEDURE FOR THE ISOLATION OF HIGH-QUALITY PHAGE-LAMBDA DNA USING A DEAE-CELLULOSE COLUMN
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DOI：
10.1016/0003-2697(88)90325-9
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发表时间：
1988-02-01
影响因子：
2.9
通讯作者：
SHERMAN, LA
中科院分区：
文献类型：
--
作者：
REDDY, KJ;KUWABARA, T;SHERMAN, LA
一种简单快速地纯化大量噬菌体波长的方法。描述了粒子和DNA。该程序包括对噬菌体颗粒进行DEAE-纤维素柱层析，并用低离子强度的缓冲液将噬菌体颗粒从柱子上洗脱。得到的噬菌体与来自大肠杆菌宿主细胞的RNA、DNA和蛋白质很好地分离。兰布达。用常规的苯酚提取和乙醇沉淀的方法从纯化的噬菌体颗粒中提取DNA。该程序不使用核酸酶、蛋白酶、洗涤剂或CsCl2密度梯度离心法。兰布达。该方法获得的DNA纯度与CsCl2密度梯度离心法制备的DNA相当，经限制性内切酶酶切、连接、放射性标记和双链DNA测序。介绍了一种从1L液体裂解液中提取0.5~1.0 mg DNA的详细方法，该方法简单、廉价、省时，特别适用于大规模分离Lambda。DNA
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, radiolabelling, 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.