文献类型： 外文期刊

作者： Nawawi, Omar ¹ ; Abdullah, Mohd Puad ² ; Yusuf, Chong Yu Lok ¹ ;

作者机构： 1.Univ Teknol MARA, Fac Plantat & Agrotechnol, Lab Plant Genet & Cell Biol, Jasin Campus, Merlimau 77300, Melaka, Malaysia

2.Univ Putra Malaysia, Fac Biotechnol & Biomol Sci, Dept Cell & Mol Biol, Upm Serdang 43400, Selangor, Malaysia

3.Yunnan Acad Agr Sci, Inst Trop & Subtrop Cash Crops, Baoshan 678000, Peoples R China

关键词： DNA cloning; pJET1.2/blunt cloning vector; pJET1.2N plasmid; Positive selection

期刊名称：3 BIOTECH （ 影响因子：2.8； 五年影响因子：3.1 ）

ISSN： 2190-572X

年卷期： 2023 年 13 卷 7 期

页码：

收录情况： SCI

摘要： Positive selection vectors carry a lethal gene encoding a toxic product that is harmful to most laboratory E. coli strains. Previously, we reported a strategy for in-house production of a commercial positive selection vector, the pJET1.2/blunt cloning vector, using common laboratory E. coli strains. However, the strategy involves lengthy gel electrophoresis and extraction procedures to purify the linearized vector after digestion. Here, we streamlined the strategy to eliminate the gel-purification step. A uniquely designed short fragment called the Nawawi fragment was inserted into the coding sequence of the lethal gene of the pJET1.2 plasmid, resulting in the pJET1.2N plasmid that can be propagated in the E. coli strain DH5a. Digestion of the pJET1.2N plasmid with EcoRV released the Nawawi fragment, and the resulting blunt-ended pJET1.2/blunt cloning vector can be used directly for DNA cloning without prior purification. Cloning of a DNA fragment was not hindered by the Nawawi fragments carried over from the digestion step. After transformation, the pJET1.2N-derived pJET1.2/blunt cloning vector produced > 98% positive clones. The streamlined strategy accelerates the in-house production of the pJET1.2/blunt cloning vector and enables DNA cloning at a lower cost.