EFFICIENT CLONING METHOD THAT SELECTS THE RECOMBINANT CLONES

Directional cloning using cohesive ends is the most efficient cloning method. However, sometimes it is necessary to use blunt ends to clone a DNA fragment into the plasmid vector. Compared with that of cohesive ends, efficiency of blunt-end ligation is low. Compared with the nati ve blunt ends (e.g., SmaI or EcoRV), blunt-end ligation is particularl y difficult when blunt ends are derived from overhangs. This results i n low efficiency of insertion and high background from self-ligation o f the vector. To remedy the problem, we developed a ''positive selecto r'' cloning strategy that provides positive selection for the recombin ant clones. It is particularly useful when making complex recombinant constructs and the choice of restriction sites is limited.