Cold Spring Harb Protoc. 2021 Apr 1;2021(4):2021/4/pdb.prot101287. doi: 10.1101/pdb.prot101287.
The following is an elegant and simple protocol for generating and cloning blunt-ended DNA. Incubation of a ligation reaction in the presence of an excess amount of restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are regenerated when plasmid molecules ligate to themselves. The method requires that ligation of the plasmid to a target DNA molecule destroys the restriction site, so preventing the restriction enzyme from digesting recombinants generated during the ligation reaction. The net effect of constant reclamation of unit-length linear vector molecules is to drive the equilibrium of the ligation reaction strongly in favor of recombinants between vector and insert. The method is efficient because regeneration of vector DNA, ligation, and polishing the termini of PCR-generated fragments of DNA all occur simultaneously in the same reaction mixture.
以下是一个优雅而简单的方案，用于生成和克隆平端 DNA。在过量限制酶存在下孵育连接反应，可以显著提高重组质粒的产量。限制酶的作用是在质粒分子自身连接时在重新生成的限制位点处切割环状和线性串联体。该方法要求质粒与靶 DNA 分子的连接破坏限制位点，从而防止限制酶消化连接反应中产生的重组体。线性载体分子的单位长度的不断再回收的净效应是强烈地驱动连接反应的平衡有利于载体和插入物之间的重组体。该方法效率高，因为载体 DNA 的再生、连接和 DNA 的 PCR 片段末端的抛光同时在同一个反应混合物中发生。
