OriCiro® Cell-Free Cloning System/PASS V4.1.2 v1

OriCiro® Cell-Free Cloning System is a rapid and powerful tool replacing cumbersome DNA cloning (plasmid construction) process relying on E. coli. The system consists of two kits. OriCiro Assembly Kit allows seamless assembly of multiple overlapping DNA fragments. The assembly product can be added directly to OriCiro Amp Kit to get selective amplification of your target circular DNA (Figure 1). The amplified product is supercoiled DNA topologically identical to plasmid DNA isolated from E. coli. 1. OriCiro Assembly Kit: Multiple DNA fragments are assembled seamlessly at 42 ̊C for 30 minutes via ~40 bp overlapping ends (Figure 2). DNA fragments generated by PCR or restriction enzyme digestion are available. Our unique enzyme-based annealing mechanism allows powerful assembly up to 50 fragments simultaneously. 2. OriCiro Amp Kit: The reaction consists of 26 purified enzymes involved in chromosome replication of E. coli. The chromosome replication cycle repeats autonomously at around 30 ̊C, enabling exponential amplification of circular DNA having oriC with extremely high fidelity (10-8 error/base/cycle) (Figure 3). The kit yields up to 1 μg circular DNA per 10 μL reaction at 33 ̊C for 6 hr. The maximum amplification size is 50 kb in the current version of the kit. n.b. OriCiro Amp NEEDS oriC Cassette (0.4 kb) which can be inserted into circular DNA using OriCiro assembly kit. References: 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 2. 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) 3. M. Su’etsugu, H. Takada, T. Katayama, H. Tsujimoto, Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle, Nucleic Acids Research, 2017, 45 (20), 11525– 11534

Polymerase chain reaction (PCR) v2

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

Polymerase chain reaction (PCR) v2

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

Polymerase chain reaction (PCR) v2

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

Polymerase chain reaction (PCR) v1

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

Polymerase chain reaction (PCR) v2

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

Polymerase chain reaction (PCR) v2

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

CONSTRUCTING A NOVEL PLASMID TO INCREASE PURITY EFFECTIVENESS FOR RECOMBINANT PROTEINS EXPRESSED IN ESCHERICHIA COLI CELLS

Nowadays, requirement of supply for recombinant proteins has increased in several fields such as food technology, medical pharmacy, clinical diagnose or environment treatment. The recombinant proteins have become the commercialized products and of that yielded with increasing in a large number per year. Besides, supposing that on extending of the his-tag of the pET111a plasmid may be facilitate for removing his-tag and more effective in protein purification. In this study, nine nucleotides (GCGGCGGCG) coding three alanine residues were added to positions followed hexa-histidine tag (his-tag) on a pET11a plasmid construction. The SDS-PAGE result of each recombinant protein contained the long-modified tag after purification process almost only exhibited single band on gel. Based on alike observed consequences for three recombinant proteins already refined the purity effectiveness reach to upper 98% in the total of existing proteins inside the solution. Hence, the novel pET11a plasmid construction could become an effective plasmid for the aim of harvesting high-purified recombinant proteins.

Protocol for examining and eliminating base editor-induced genome-wide and transcriptome-wide off-target mutations

Fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications but suffers from off-target (OT) mutations. Here, we applied a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. This step-by-step protocol describes the plasmid construction of tBE system, determination of genome/transcriptome-wide OT mutations and tBE-mediated base editing in vivo.