两种pUC18高效T载体的构建

T载体是用于直接克隆PCR产物的线性载体.在此之前,克隆PCR片段时一般先用Klenow片段酶或T4DNA聚合酶削平PCR产物两端,克隆过程中又大都不能使用碱性磷酸酶为载体片段脱磷,因为绝大多数PCR引物5’端未磷酸化,T载体的诞生使分子生物学工作者摆脱了这一窘境,而且,T载体的3’端突出的T碱基与PCR产物3’端由于Taq酶非模板依赖的末端转移酶活性而添加的A碱基1]互补,使载体与PCR产物的连接效率大大提高.由于具有上述优点,T载体从一产生就引起人们极大的兴趣,很多公司也相继推出了各自的T载体系统,并运用该技术改造了很多传统载体.本…

Construction of Two Highly Efficient pUC18 T-vectors

Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3'T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm Ⅰ recognition sites were introduced to both ends of the PCR product.This fragment was cloned into the Sma Ⅰ site of pUC18 or the linearized pUC18 with its polylinker deleted.The recombinant plamids were cleaved with Xcm Ⅰ.the larger fragments generated which had 3'T over hangs at both ends were used as T vctors.Genes of rotavirus VP7 and the plasminogen k5 were successfully cloned into these two T vectors with recombination efficiency (recombinants/transformants×100%) of 100%,no blue/white clolny screening assay was needed.