Subcloning of a gene or DNA fragment into an appropriate vector is an essential technique that is widely used in current molecular biology. Major advantages include: (1) DNA fragments of interest can be subcloned into a high-copy number of plasmid vectors and be amplified up to 300-fold by plasmid replication in E. coli, (2) vectors used for subcloning usually contain SP6, T7 or T3 promoters upstream from the polycloning sites, which allows preparing sense RNA or antisense RNA of the inserted cDNA as hybridization probes, (3) the SP6, T7 and T3 promoters can enable an investigator to sequence the DNA insert on both strands in opposite directions, and (4) DNA constructs can be prepared by inserting a gene or cDNA of interest into an appropriate vector for gene transfer and expression in cultured cells and animals.

The question is what the principles are and how to insert a gene or DNA fragment of interest into the right place in a vector. The general procedures and principles start with restriction enzyme digestion of vector and insert DNA. Following digestion, sticky or blunt ends are generated. Compatible sticky ends of vectors and DNA inserts will be ligated together using T4 DNA ligase, forming recombined constructs. In case of incompatible ends between vectors and inserts, the ends can be blunt-ended by 5' or 3' end filling. DNA constructs can be made by blunt-end ligation. The constructs are then transformed into an appropriate bacterial strain in which plasmids are replicated up to 300 copies per cell. Transformed bacterial colonies will be selected using appropriate antibiotics. This chapter describes the well-established, detailed methods routinely used in our laboratories.

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