We have developed a method to clone DNA fragments into the E. coli plasmid vectors with almost 100% efficiency (Goto and Nagano, 2013). This method is based on highly efficient yeast-based in vivo cloning, and the subsequent cloning of the constructed plasmids into E. coli. Our method is useful for various applications: multifragment DNA cloning, cloning of large DNA fragments, and cloning into large plasmid vectors. Furthermore, the sites at which DNA fragments are joined are not always located at the restriction ends in the plasmid vector, thus making the cloning method more flexible. Our system does not require manipulation for assembling or joining DNA fragments in a test tube, the efficiency of which may sometimes depend on the reaction conditions or the skills of the person performing the procedure. Therefore, both success rate and efficiency are extremely high. However, our system has a disadvantage in that it requires 2 steps for transformation. Our method is an improved version of previously developed methods (Iizasa and Nagano, 2006; Nagano et al., 2007). Next figure shows the flowchart of our method.
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