A modified plasmid vector pCMV-3Tag-LIC for rapid, reliable, ligation-independent cloning of polymerase chain reaction products
A modified plasmid vector pCMV-3Tag-LIC for rapid, reliable, ligation-independent cloning of polymerase chain reaction products
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改良的质粒载体 pCMV-3Tag-LIC，用于快速、可靠、不依赖于连接的聚合酶链式反应产物克隆
DOI：
10.1016/j.ab.2010.08.042
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发表时间：
2011-01-15
影响因子：
2.9
通讯作者：
Zhang, Xiaodong
中科院分区：
文献类型：
--
作者：
Du, Runlei;Li, Shangze;Zhang, Xiaodong
在这里，我们提出了一个修改后的载体pCMV-3 Taq-LIC的一个快速，简单，相对便宜的方法来构建表达构建体。经Nt.BspQI和EcoRV双酶切后，获得了一个具有特异性11个碱基单突出端的线性载体。具有互补突出端的聚合酶链式反应（PCR）产物通过在引物中构建适当的延伸并用T4 DNA聚合酶处理它们来产生。插入片段和载体的退火在不存在连接酶的情况下通过简单混合DNA片段进行。这个过程是非常具体的，因为只有所需的产品可以形成。利用该载体成功构建了hnRNPK全长互补DNA（cDNA）表达质粒。(C)2010年爱思唯尔公司All rights reserved.
Here we present a modified vector pCMV-3Taq-LIC for a rapid, simple, and relatively cheap method to build expression constructs. After being digested by Nt.BspQI and EcoRV, a lineal vector with specific 11-base single overhangs is obtained. Polymerase chain reaction (PCR) products with complementary overhangs are created by building appropriate extensions into the primers and treating them with T4 DNA polymerase. The annealing of the insert and the vector is performed in the absence of ligase by simple mixing of the DNA fragments. This process is very specific because only the desired products can form. Using this vector, we successfully constructed hnRNP K full-length complementary DNA (cDNA) expression plasmid. (C) 2010 Elsevier Inc. All rights reserved.