构建丝状真菌大片段基因组文库载体DNA的制备

Preparation of Vector DNA for Filamentous Fungal Large-insert Genomic Library Construction

载体DNA的制备是构建大片段基因组文库的关键步骤之一。高质量载体DNA受到酶切、脱磷等因素的影响。以载体pBHYG为材料 ,优化了限制性内切酶HindIII酶切和小牛肠碱性磷酸酶 (CIAP)脱磷的作用条件 ,并在T4连接酶作用下自连 ,通过胶回收纯化制备了可用于进一步构建大片段基因组文库的线性载体DNA。

Preparation of vector DNA is a crucial step to large-insert genomic library construction.Preparation of highly purified vector DNA is affected by a series of factors such as digestion of restriction enzyme and dephosphorylation of linearized vector DNA.In our study,conditions of vector pBHYG digested by restriction enzyme of Hind III and dephosphorylated by calf intestine alkaline phosphatase (CIAP) were optimized respectively.Self ligation under T4 ligase of digested/dephosphorylated vector DNA and gel purification were conducted to get high quality linearied vector DNA for construction of large-insert genome library.