Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

BACKGROUND: The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs. RESULTS: We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector. CONCLUSION: The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.