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and fragments with 3’-A overhang, the Zero Background Omni Topoisomerase Cloning Kit utilizes DNA strand transfer activity of Viccinia virus topoisomerase I for the quick cloning of DNA fragments up to 10 kb. 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 (refer to the illustration below). This transferring reaction is rapid and reproducible. The cloning vector pCloneEZ-Omni-Amp/HC and pCloneEZ-Omni-Kan/HC included in this kit are high copy number plasmid engineered to tolerate mild toxic genes. For fast cloning of extremely toxic genes please chose our advanced CloneRanger Topoisomerase Kit.
Does not include competent cell.
Zero Background: >90% of randomly picked clones contain the right insert.
Fast: Instant transformation after mixing PCR fragment with vector, no incubation needeed.
CloneSmarter™ Zero Background Blunt Topoisomerase Cloning Kit Flow Chart:
|Components||C5865-25/C5866-25 (25 rxns)||C5865-50/C5866-50 (50 rxns)|
pCloneEZ-Omni-Kan/HC (20 ng/μl)
|25 µl||50 µl|
|10X Enhancer||30 µl||60 µl|
|Control Insert lacZα (20ng/μl)||10 µl||10 µl|
|Blank Control Vector (100ng/μl)||20 µl||20 µl|
|M13F Sequencing Primer (10pmol)||100 µl||100 µl|
|M13R Sequencing Primer (10pmol)||100 µl||100 µl|