Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5
Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5
复制标题
大细菌质粒的分离以及 P2 不相容组质粒 pMG1 和 pMG5 的表征
作者：
J. Hansen;R. H. Olsen
来自根癌农杆菌、鼠伤寒沙门氏菌、大肠杆菌、恶臭假单胞菌和铜绿假单胞菌的大质粒使用不需要超离心但包括设计用于将大质粒DNA与细菌折叠染色体分离的步骤的程序常规且一致地分离。它还选择性地去除断裂染色体的片段。琼脂糖凝胶电泳可以很容易地观察到各种大质粒，包括5个大小在70和85兆道尔顿（Mdal）之间，6个在90和143 Mdal之间，一个大于200 Mdal，一个大于300 Mdal。该分离程序允许对两种IncP 2质粒pMG 1和pMG 5的分子大小进行初步估计，其分别为312和280 Mdal。通过凝胶电泳进行大小测定的标准曲线，包括大小在23和143 Mdal之间的质粒，对于300 Mdal大小范围的质粒不能线性外推。不同质粒对分离过程的独特反应包括IncP 1质粒对高pH的敏感性和20-Mdal“隐蔽”质粒的共同分离。
Large plasmids from Agrobacterium tumefaciens, Salmonella typhimurium, Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa were routinely and consistently isolated using a procedure which does not require ultracentrifugation but includes steps designed to separate large-plasmid DNA from the bacterial folded chromosome. It also selectively removes fragments of broken chromosome. A variety of large plasmids was readily visualized with agarose gel electorphoresis, including five between 70 and 85 megadaltons (Mdal) in size, six between 90 and 143 Mdal, one that was larger than 200 Mdal, and one that was larger than 300 Mdal. This isolation procedure allowed initial estimation of the molecular sizes of the two IncP2 plasmids, pMG1 and pMG5, which were 312 and 280 Mdal, respectively. A standard curve for size determination by gel electrophoresis including plasmids between 23 and 143 Mdal in size did not extrapolate linearly for plasmids of the 300-Mdal size range. Unique response of different plasmids to the isolation procedure included sensitivity of IncP1 plasmids to high pH and the co-isolation of a 20-Mdal "cryptic" plasmid in conjunction.