Overlap extension cloning of PCR products into a Gateway-compatible plasmid vector.

IF 2.2 4区工程技术 Q3 BIOCHEMICAL RESEARCH METHODS

BioTechniques Pub Date : 2023-07-01 DOI:10.2144/btn-2023-0001

Omid Zare-Mehrjerdi, Gracie Trader, Viktor Kirik

引用次数: 0

Abstract

A PCR cloning method that combines a dual selection pGATE-1 plasmid vector and an improved overlap extension cloning was developed. This efficient and cost-effective method allows for the introduction of DNA fragments into the Gateway cloning pipeline. The cloning efficiency is facilitated by a dual selection that includes the ccdB gene and gentamicin resistance. For users of the Gateway cloning system, substantial cost saving comes from eliminating BP recombination and ligation reactions to introduce DNA fragments into pDONR or pENTR vectors. Beyond the Gateway technology, this recombination-based cloning system can be used to efficiently clone PCR amplicons by adding 24-base pair adaptor sequences that are utilized by bacterial homologous recombination mechanism.

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PCR产物在gateway相容质粒载体上的重叠延伸克隆。

建立了一种双选择pGATE-1质粒载体和改进的重叠延伸克隆相结合的PCR克隆方法。这种高效和经济的方法允许将DNA片段引入到Gateway克隆管道中。ccdB基因和庆大霉素耐药性的双重选择促进了克隆效率。对于Gateway克隆系统的用户来说，省去了将DNA片段导入pDONR或pENTR载体的BP重组和连接反应，节省了大量成本。除了Gateway技术之外，该基于重组的克隆系统可以通过添加细菌同源重组机制所使用的24碱基对适配器序列来高效地克隆PCR扩增子。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

BioTechniques 工程技术-生化研究方法

CiteScore

4.40

自引率

0.00%

发文量

审稿时长

3.3 months

期刊介绍： BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies. Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers: Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices Surveys of technical approaches related to broad fields of research Reviews discussing advancements in techniques and methods related to broad fields of research Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.