利用XcmⅠ构建T载体

Construction of T-vector by XcmⅠ

目的 :构建能自我复制的T载体。方法 :设计一对引物 ,引入XcmⅠ酶切位点 ,通过PCR方法用PET 2 8(a) +作模板扩增5 5 0bp ,插入Pa载体后用限制性内切酶、PCR及DNA测序等方法作鉴定。结果 :用XcmⅠ酶切可见 5 40bp及 3810bp双酶切条带 ,用此载体作模板可扩增出 5 5 0bp片段 ,测序结果与预期序列一致。结论 :带有两个XcmⅠ位点的环状载体被成功构建。

Objective To construct T-vector with characteristics of replicating itself.Methods By designing two primers on the site of XcmⅠ,550bp fragment was amplified by polymerase chain reaction(PCR) using template of PET28(a) +;the plasmid with inserted fragment was identified by methods of restrictive analysis,PCR and DNA sequencing.Results Two bands of 540bp and 3810bp appeared by XcmⅠ;550bp fragment can be amplified by PCR;and sequencing result was in accordance with the expected sequence.Conclusion A circular vector with two XcmⅠ sites has been successfully constructed.