重组工程
同源重组
质粒
原噬菌体
转化(遗传学)
遗传学
生物
染色体
体外重组
DNA
细菌人工染色体
细菌圆形染色体
克隆(编程)
重组
计算生物学
噬菌体
大肠杆菌
分子克隆
基因
基因组
DNA复制
计算机科学
肽序列
程序设计语言
作者
Lynn C. Thomason,Donald L. Court,Mikail Bubunenko,Nina Costantino,Helen Rose Wilson,Simanti Datta,Amos B. Oppenheim
标识
DOI:10.1002/0471142727.mb0116s78
摘要
Abstract The bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination functions efficiently to recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. Support protocols describe a two‐step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a co‐electroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.
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