一种高效低背景T载体的构建

Construction of the High Efficiency and Low Background T Vector

介绍一种构建高效低背景T载体的通用方法。使用含有ccdB致死基因的gateway cassette片段作为插入DNA片段以降低背景干扰,连接到pGEM-T easy载体骨架上,通过内切酶XcmI酶切重组质粒即得到T载体。对重组质粒进行了酶切,PCR和测序验证,并且利用连接效率实验证实了T载体具有100%的阳性克隆率。构建的T载体不仅继承了pGEM-T easy的众多优点,而且具有高效、低背景的卓越特点;另外,引入的常用限制性内切酶和LR重组反应介导的gateway技术为亚克隆提供了便利。

A universal method of the high efficiency and low background T vector construction was developed.The gateway cassette fragment containing the ccdB killer gene,used as medium DNA,was ligated to the vector backbone pGEM-T easy,and then the recombinant plasmid was digested by XcmI restriction enzyme to produce the T vector.The recombinant plasmid was confirmed by digestion pattern,PCR identification and sequencing,and the T vector was proved to be high efficiency and low background.This T vector had not only many advantages of pGEM-T easy vector,but also had very high efficiency and low background for molecular cloning.What＇s more,several restriction enzymes introduced and gateway technology with LR recombination reaction provided convenience for insert fragment subcloning.