Jeung Ji Ung, Cho Sung Ki, Shim Kyu Suk, Ok Sung Han, Lim Dae Sik, Shin Jeong Sheop
Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea.
Plasmid. 2002 Sep;48(2):160-3. doi: 10.1016/s0147-619x(02)00122-1.
For applications such as sequencing, transfection, and in vitro transcription, PCR products have to be subcloned into plasmids. Many strategies are used for cloning, blunt-end ligation or the incorporation of restriction endonuclease sites into PCR primers for appropriate vectors. However, the most convenient and direct method is T/A cloning. In this study, we developed two of the pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites, and these T vectors have all the features of pGEM-7Zf(+): f1 ori, T7, and SP6 RNA polymerase promoters, the alpha-peptide coding region of beta-galactosidase for X-gal blue/white color selection, the beta-lactamase gene for recombinant colony selection, and binding sites for pUC/M13 forward and reverse sequencing primers. These AhdI-containing phagemid vectors, pGEM-NJ105 and pGEM-NJ107, are useful for the easy and inexpensive preparation of T vectors and direct cloning of PCR products.
对于诸如测序、转染和体外转录等应用，PCR产物必须亚克隆到质粒中。许多策略用于克隆，如平端连接或将限制性内切酶位点引入用于合适载体的PCR引物中。然而，最方便直接的方法是T/A克隆。在本研究中，我们利用AhdI限制性内切酶位点构建了两种pGEM-7Zf(+)噬菌粒T尾载体，这些T载体具备pGEM-7Zf(+)的所有特征：f1 ori、T7和SP6 RNA聚合酶启动子、用于X-gal蓝/白颜色筛选的β-半乳糖苷酶α-肽编码区、用于重组菌落筛选的β-内酰胺酶基因以及pUC/M13正向和反向测序引物的结合位点。这些含AhdI的噬菌粒载体pGEM-NJ105和pGEM-NJ107，有助于简便且低成本地制备T载体以及直接克隆PCR产物。
