|pTOPO-Blunt Simple Vector(30ng/ul)||20 ul||80 ul|
|1000bp Control（30ng/ul）||5 ul||5 ul|
|10 ´ Enhancer||20 ul||80 ul|
Note: All reagents, when store in -20 °C, are stable for 12 months
The Zero Background Blunt Simple Topoisomerase Cloning Kit is designed for fast cloning of blunt ended DNA fragments up to 10 kb generated by high fidelity DNA polymerase such as KOD, Pfu and Phusion ext. DNA fragments obtained by restriction digestion or mechanical shearing can also be cloned after end polishing to become blunt ended. It utilizes DNA strand transfer activity of Viccinia virus topoisomerase I. Vaccinia virus DNA topoisomerase I forms a 3’-phosphoryl intermediate with the plasmid vector containing 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 Simple included in this kit are high copy number plasmids engineered to tolerate mild toxic genes. Regions flanking the cloning site of pTOPO-Blunt Simple vectors do not contain any common restriction sites which eliminates possible redundancy of restriction sites.
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