UcallM® C2×HiFi Gibson Assemble Mix

Gibson assembly enables isothermal seamless cloning: multiple DNA fragments are directionally assembled into linearized vectors with zero extra base residues. UcallM® 2×HiFi Gibson Assemble Mix delivers high-fidelity, robust DNA ligation for library construction and protein-expression-vector assembly. The mix contains T5 exonuclease (generates 3'-overhangs), high-fidelity DNA polymerase (gap-filling) and Taq DNA ligase (nick-sealing).

Experimental Workflow

Figure 1-71. Schematic diagram of Gibson assembly principle. A) Working mechanism of the three enzymes used in Gibson assembly; B) Assembly of a single insert fragment; C) Assembly of multiple insert fragments. Colored blocks indicate homology arms, and the annealing temperature should be higher than 48 °C. The reaction system for Gibson assembly contains T5 exonuclease, high-fidelity DNA polymerase, and Taq DNA ligase. T5 exonuclease exhibits potent 5′→3′ exonuclease activity. It digests cloned fragments to generate 3′ overhang sticky ends, and complementary base pairing occurs between homologous termini. Subsequently, high-fidelity DNA polymerase fills the gaps in the 5′→3′ direction. Taq DNA ligase catalyzes the formation of phosphodiester bonds between the 5′-phosphate and 3′-hydroxyl groups at nicked sites.

Key Advantages 1.Cloning accuracy > 95 % 2.Stable liquid master-mix format minimizing freeze-thaw-induced activity loss 3.High assembly efficiency, suitable for large-capacity DNA-library construction Applications 1.DNA fragment assembly 2.Molecular cloning 3.Protein-expression-vector building 4.Site-directed mutagenesis 5.Small-genome reconstruction Measured Data

Figure 1-72. Measured performance of this kit. A) LB plate image after transformation of reaction products into competent E. coli DH5α, including single 800 bp insert assembly and multi-fragment assembly with 800 bp and 500 bp inserts; B) Colony PCR identification of recombinant clones, all clones tested were positive.

Experimental Notes 1.Recommended insert-to-vector molar ratio = 1 : 1 ~ 1 : 2 2.Homology-arm length: 15-40 bp; primer Tm ≥ 50 °C 3.Linearize vector by PCR; digest template DNA with Dpn I to suppress background 4.For single-fragment cloning, PCR products can be added directly without purification; PCR-product volume shall not exceed 20 % of total reaction volume 5.For multi-fragment assembly, gel-purification of inserts and vector is strongly recommended. Customer Publication Nat Microbiol. How KorB transforms from a DNA-sliding clamp into a long-range transcriptional silencer (DOI:10.1038/s41564-024-01915-3).

Figure 1-73. In this study, Gibson assembly was used for DNA fragment ligation during plasmid construction. Multiple plasmids were generated for subsequent protein expression and functional studies.