|pTOPO-T 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 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 included in this kit are high copy number plasmids engineered to tolerate mild toxic genes. Regions flanking the cloning site of pTOPO-TA vectors have multiple common restriction sites for release of the cloned fragment by single or double restriction digestion.
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