目录摘要..................................................................................................................1引言......................................................................................................................11.材料与方法.....................................................................................................41.1实验材料..................................................................................................41.1.1菌株...............................................................................................41.1.2体质粒载.......................................................................................41.1.2酶和试剂.......................................................................................41.1.2.1酶........................................................................................41.1.2.2抗生素................................................................................51.1.2.3生化试剂............................................................................51.2仪器设备..................................................................................................51.3实验方法..................................................................................................51.3.1酶切...............................................................................................51.3.2DNA片段补平..............................................................................61.3.3大片段去磷酸化.............................................................................1.3.4乙醇沉淀纯化DNA.....................................................................71.3.5胶回收...........................................................................................71.3.6连接.................................................................................................1.3.7KODplus高保真酶PCR反应...............................................................1.3.8检测PCR反应.............................................................................81.3.9Gateway技术..................................................................................1.3.9.1BP反应...............................................................................71.3.10大肠杆菌转化...............................................................................1.3.11质粒提取.....................................................................................92.结果与分析......................................................................................................122.1pWBVec8+Ga-b载体的酶切.................................................................122.2载体去磷酸化........................................................................................122.3载体补平................................................................................................122.4Cas9质粒的酶切....................................................................................132.5含有Cas9元件片段的补平..................................................................132.6Cas9元件片段与载体的连接................................................................132.7pWBVec8+Ga-b-CRISPR质粒检测......................................................132.8入门载体的构建.......................................................................
