Methodology
Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
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* Corresponding author: Tobias Fromme fromme@staff.uni-marburg.de
Department of Animal Physiology, Faculty of Biology, Philipps-University, D-35043 Marburg, Germany
Journal of Biological Engineering 2007, 1:7 doi:10.1186/1754-1611-1-7
Published: 26 November 2007Abstract
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.