Cold Spring Harb Protoc. 2020 Apr 1;2020(4):098053. doi: 10.1101/pdb.prot098053.
This protocol describes the preparation of the shuttle vector before its introduction into bacterial artificial chromosome (BAC) host cells for BAC two-step engineering. The homology arm sequences, prepared previously, are introduced by ligation into the digested shuttle vector DNA to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector.
本方案描述了穿梭载体在引入细菌人工染色体 (BAC) 宿主细胞进行 BAC 两步工程之前的准备工作。之前制备的同源臂序列通过连接引入到经酶切的穿梭载体 DNA 中，为 BAC 克隆内的重组提供位点。单个细菌转化体的粗裂解物可作为聚合酶链反应 (PCR) 分析的模板，以确认重组穿梭载体中同源臂的存在。
