基于XcmⅠ酶切的高效TA克隆载体的构建

目的：制备基于XcmⅠ酶切的高效TA克隆载体，并检测其克隆PCR产物的效果。方法：设计一对互补配对的寡核苷酸，经过变性及退火后插入质粒pUC19的多克隆位点，从而在该多克隆位点中引入2个XcmⅠ酶切位点，用XcmⅠ酶切后即获得含有3’突出T碱基的T载体；为了提高该T载体的克隆效率，优化了2个XcmⅠ酶切位点之间的碱基数目，排除了载体自连产生白色克隆的可能性，使假阳性大大减少；此外，为了便于完全酶切与未完全酶切载体的分离，在2个XcmⅠ之间插入了一段无关DNA片段。结果：改进得到的T载体可以有效克隆PCR产物，其阳性克隆率可达95％。结论：构建了基于XcmⅠ酶切的TA克隆载体，经过改进的T载体具有很高的克隆效率。

Construction of Xcm Ⅰ-Based,Highly Efficient TA Cloning Vectors

Objective： To construct Xcm Ⅰ -based TA cloning vectors and test their effectiveness in cloning of PCR products. Methods： Two complementary oligonucleotides were synthesized. After denaturation, the two oligonucleotides were annealed and cloned into the multiple cloning sites（MCS） of plasmid pUC19. In this way, two Xcm I sites were introduced into the pUC19 MCS. This plasmid was digested with Xcm I to derive a T-vector with a 3 overhang T base. To increase the efficacy of the above vector, the sequence between the two Xcm Ⅰ sites was adjusted to exclude the possibility of white colonies generated by self-ligation of the vector and thus greatly reduce the percentage of the false positive white clones. Furthermore, to enhance the separation of the fully digested T-vector DNA molecules from the partially digested or un-digested vector molecules, a DNA fragment was inserted into the plasmid between the two Xcm I sites. Results： The T-vectors constructed were efficient in cloning PCR products, with an improved T-vector achieving a cloning efficiency of 95%. Conclusion： The Xcm I -based TA cloning vectors were constructed, and the improved versions of the T-vector demonstrated very high cloning efficiency.