Tris-HCl, Molecular Biology Grade

公司名称: Promega

产品编号: H5123

Similar Protocol

One Plate-double Nutrient Endospore Activation Method

Bhagwan Narayan Rekadwad, Juan M. Gonzalez and Chandrahasya N. Khobragade

Published online: 2020-01-05

Bio-protocol()

Company-protocol()

Other protocol()

Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome

Author:

Analise Z Reeves and Cammie F Lesser,

Date:

2016-11-20

[Abstract] Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. ... [摘要] 由于其大尺寸，细菌致病性岛和其它连续操纵子可能难以使用常规方法克隆。在这里我们描述了一个强大的3步法从毒力质粒或粘粒转移大型定义的片段到更小的自主复制质粒或直接到细胞染色体，并入内源性酵母和λ红色同源重组系统的定义网站。该方法已经成功地用于分离和整合至少31kb的连续DNA，并且可以容易地适应于E的重组。大肠杆菌及其近亲。

[背景] 分离和繁殖大片DNA的能力大大扩展了基因网络和操纵子的研究。然而，用于该目的的传统使用的工程质粒，例如细菌人工染色体（BAC），虽然极其有用，但是受到DNA稳定性，拷贝数和复杂装配要求的问题的限制。或者，将构建体直接并入细菌染色体中通过减少由于存在多个基因拷贝引起的基因表达的变化以及确保基因的稳定维持而提供了优点，同时还避免了对抗生素选择的需要。这里描述的方法最初被设计为捕获和转移编码Shigella flexneri 3型分泌系统的31kb DNA操纵子到大肠杆菌染色体上（Reeves ...

产品评论

Tris-HCl, Molecular Biology Grade

Tris-HCl, Molecular Biology Grade

免责声明