Note: All reagents, when store at -20 °C, are stable for 12 months.

Description

The Zero Background Blunt Topoisomerase Cloning Kit is designed to fast-cover blunt-ended DNA fragments up to 10 kb generated by high-fidelity DNA polymerases such as KOD, Pfu and Phusion. DNA fragments obtained by restriction digestion or mechanical shearing can also be cloned after end polishing to become blunt-ended. It utilizes the DNA strand transfer activity of Vaccinia virus topoisomerase I. Vaccinia virus DNA topoisomerase I form a 3’-phosphoryl intermediate with the plasmid vector containing a cleavage recognition motif of 5’CCCTT↓. Covalently bound topoisomerase I then transfer the incised vector DNA strand to the DNA fragment to be cloned with free 5’-OH terminuses. This transferring reaction is rapid and reproducible. The cloning vector pTOPO-Blunt in this kit are high-copy number plasmids engineered to tolerate mild toxic genes. Regions flanking the cloning site of pTOPO-Blunt vectors have multiple common restriction sites for releasing the cloned fragment by single or double restriction digestion.