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