Abstract
Conventional subcloning into plasmid vectors often involves dephosphorylation, gel electrophoresis, DNA extraction, and purification to isolate the target insert and the cleaved plasmid. This is not only time-consuming but very often problematic. We have developed strategies that can circumvent these steps by mixing digested donor and recipient plasmids together for ligation. These strategies capitalizes on: (1) the ability to enhance the ligation efficiency of desired DNA fragments into the target vector by the generation and removal of small (<50 bp) fragments from nontarget DNA using peripheral restriction sites and spin column technology and (2) the elimination of undesired ligation products after ligation by using the Lac Z gene, differences in antibiotic resistance among plasmid vectors, and unique restriction sites situated in nontarget DNA fragments.
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Xu, Q., Zhang, D. & Downie, B. Rapid and efficient subcloning of DNA without dephosphorylation or gel electrophoresis. Mol Biotechnol 29, 111–117 (2005). https://doi.org/10.1385/MB:29:2:111
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DOI: https://doi.org/10.1385/MB:29:2:111