介绍一种PCR产物克隆的新方法——T-A克隆法. 用改良碱裂解法抽提pBluescript SK(+)质粒,用EcoRⅤ将pBluescript SK(+)质粒切成平端,利用Taq DNA聚合酶及dTTP制备pBluescript SK(+) T-A载体. 将β-actin cDNA片段克隆入自制的pBluescript SK(+) T-A载体,经EcoRⅠ及HindⅢ双酶切得到与设定长度一致的β-actin cDNA片段.
A new method for PCR products cloning——T-A cloning technique is introduced. pBluescript SK(+) plasmid was extracted by modified alkali lysis method. A blunt ends was generated using restriction enzyme EcoRⅤ, then T-A cloning vector was prepared in the presence of Taq DNA polymerase and dTTP. β-actin cDNA fragment was cloned into the above T-A cloning vector. After the recombinant plasmids were digested by EcoRⅠ and HindⅢ, an expected size of β-actin cDNA fragment was obtained.
李新波,赵小松,田德志,朱依纯,姚泰.一种PCR产物克隆的新方法——T-A克隆法[J].生物化学与生物物理进展,1999,26(2):187-189
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