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* Product belong to a Not for profit entity only *1

* Research use only

*1 Academic organization, a university or another institution of higher education or any nonprofit scientific or educational organization including government agencies

 Description

OriCiroTM Cell-Free Cloning System is an innovative tool enabling cell-free assembly and amplification of circular DNA molecules without E. coli transformation and culture. This product can not only streamline your workflow dramatically but also widen the scope of your genetic engineering techniques. This product is composed of OriCiro Assembly Kit and OriCiro Amp Kit and designed to produce the maximum effect when used in combination although each kit can be used independently.

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Application

• Artificially designed viral vector and plasmid construction

• Amplification of long DNA and sequences difficult to amplify by PCR

• Alternative to time-consuming and laborious  E. coli cloning

• Recombinant phage production

• Efficient cloning of any DNA sequence, including cytotoxic and GC-rich

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OriCiro Assembly

DNA fragments are assembled via overlapping ends having homologous sequences. First, each end of the fragments is digested by exonuclease to expose a single stranded homologous sequence. Then, corresponding fragments having the homologous sequence each other are annealed by enzymes.

• Efficient ligation by isothermal enzymatic reactions.

• Up to 50 DNA fragments can be assembled together as designed.

• Up to 50 kb DNA fragment can be assembled

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OriCiro Amplification(Amp)

26 kinds of purified enzymes that are essential for the E. coli chromosome replication cycle.

This Kit enables to amplify exponentially circular DNA having oriC (E. coli chromosomal origin) in an isothermal condition even from a single template molecule. Up to 1ug circular DNA can be yielded as supercoiled form per 10 ul reaction.

• More than 1 billion times amplification from 1 circular DNA molecule by isothermal reaction for several hours

• Up to 10,000 times more accurate amplification than PCR (Taq polymerase)

• Sequences with GC-rich or cytotoxic which are difficult to amplify by PCR or E. coli can be amplified.

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Reference:

1. T. Mukai, T. Yoneji, K. Yamada, H. Fujita, S. Nara, M. Su'etsugu, Overcoming the Challenges of Megabase-Sized Plasmid Construction

in Escherichia coli, ACS Synthetic Biology, 2020, 9 (6), 1315-1327

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2. M. Su’etsugu, H. Takada, T. Katayama, H. Tsujimoto, Exponential propagation of large circular DNA by reconstitution of a chromosomereplication cycle, Nucleic Acids Research, 2017, 45 (20), 11525–11534

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3. T. Hasebe, K. Narita, S. Hidaka, M. Su'etsugu, Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of

Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction. Life, 2018, 8 (43)