|pTOPO-T 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 at -20 °C, are stable for 12 months.
The Zero Background TA Simple Topoisomerase Cloning Kit is designed for the fast cloning of DNA fragments up to 10 kb generated by Taq DNA polymerase, which introduces a single 3’-A overhang to the end of the DNA product. DNA fragments generated by restriction digestion or mechanical shearing can also be effectively cloned after end polishing using Taq DNA polymerase. 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. This kit’s cloning vector pTOPO-TA Simple is a high-copy number plasmid engineered to tolerate mild toxic genes. Regions flanking the cloning site of pTOPO-TA Simple vectors do not contain any common restriction sites, eliminating the possible redundancy of restriction sites.
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