Cold Spring Harb Protoc. 2020 Nov 2;2020(11):2020/11/pdb.prot101246. doi: 10.1101/pdb.prot101246.
This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of "correct" ligation products when cloning blunt-ended target fragments, the two components of DNA in the ligation reaction must be present at an appropriate ratio. If the molar ratio of plasmid vector to target DNA is too high, then the ligation reaction may generate an undesirable number of circular empty plasmids, both monomeric and polymeric; if too low, the ligation reaction may generate an excess of linear and circular homopolymers and heteropolymers of varying sizes, orientations, and compositions. For this reason, the orientation of the foreign DNA and the number of inserts in each recombinant clone must always be validated by restriction endonuclease mapping or some other means.
本方案描述了将平头 DNA 片段克隆到线性化质粒载体中的步骤。为了在克隆平头靶片段时获得最大数量的“正确”连接产物，连接反应中的两个 DNA 组分必须以适当的比例存在。如果质粒载体与靶 DNA 的摩尔比过高，则连接反应可能会产生数量过多的、无目的的空质粒单体和多聚体；如果比例过低，则连接反应可能会产生过多的线性和环状同聚物和异聚物，其大小、方向和组成各不相同。因此，外源 DNA 的方向和每个重组克隆中的插入物数量必须始终通过限制性内切酶图谱分析或其他方法进行验证。
