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| Product Name | Gibson Assembly Ultra Master Mix |
| Catalog No. | NATR-HMM-0133 |
| Description | A 2× master mix containing all components required for the seamless assembly of multiple overlapping DNA fragments in a single isothermal reaction. The reagent combines three enzymatic activities — exonuclease, DNA polymerase, and DNA ligase — to join DNA fragments with 15–40 bp overlapping ends without the need for restriction enzymes. |
| Intended Use | Seamless assembly of multiple DNA fragments into any vector for gene cloning, pathway construction, synthetic biology applications, and multi-fragment construct assembly; also suitable for site-directed mutagenesis with overlapping primers and generation of fusion protein constructs. |
| Principle / Technology | The master mix contains a 5ʹ-exonuclease that creates single-stranded 3ʹ-overhangs on adjacent DNA fragments with complementary overlap sequences. Complementary overhangs anneal, a high-fidelity DNA polymerase fills any resulting gaps, and a thermostable DNA ligase covalently joins the assembled fragments. The three enzymes function harmoniously at 50°C in a single isothermal incubation. |
| Detection Method | Transformation of assembly product into competent E. coli; colony counting for assembly efficiency; colony PCR, restriction digestion, and Sanger sequencing of recombinant clones for accuracy verification; whole-plasmid sequencing for large construct confirmation |
| Sample Type | Linearized vector DNA and insert DNA fragments with 15–40 bp of overlapping homology; PCR products, restriction fragments, synthetic dsDNA fragments (gBlocks); fragments from 100 bp to 10+ kb; up to 15 fragments assembled simultaneously |
| Performance Range / Specifications | Assembly time: 15–60 minutes at 50°C; fragment capacity: 2–6 fragments (standard), up to 15 fragments (optimized protocol); overlap length: 15–25 bp (standard), 25–40 bp (large or multi-fragment assemblies); assembly efficiency: >95% correct clones for 2-fragment, >80% for 4-fragment assemblies; total construct size up to 100+ kb |
| Sensitivity / LOD | Assembly from as little as 10 ng per fragment with optimal overlap length; reliably detects single recombinant colony per 10 pg of correctly assembled product |
| Specificity | Seamless, scarless assembly with no extraneous sequences at junctions; >98% sequence accuracy across junctions; no restriction site or recombination site requirements, enabling sequence-independent fragment joining |
| Reaction Conditions / Protocol | Design overlapping PCR primers with 15–25 bp homology between adjacent fragments, amplify and purify each fragment, mix equimolar amounts (0.02–0.5 pmol each fragment), add 10 μL 2× master mix, water to 20 μL total, incubate at 50°C for 15–60 minutes (15 min for 2–3 fragments, 60 min for 4–6 fragments), transform 2 μL into competent E. coli, plate on selective medium |
| Components / Formulation | 2× Gibson Assembly Ultra Master Mix: 5ʹ-exonuclease, high-fidelity DNA polymerase, thermostable DNA ligase, dNTPs, NAD+, MgCl2, Tris-HCl, PEG 8000, DTT in an optimized isothermal buffer; positive control: 2-fragment assembly components |
| Storage Conditions | –20°C for long-term storage; stable on ice during use for up to 8 hours; avoid exposure to temperatures >4°C for extended periods; aliquot to minimize freeze-thaw cycles (≤5) |
| Shelf Life | 18 months at –20°C from date of manufacture |
| Package Specifications | 10 reactions, 50 reactions, and 250 reactions (based on 20 μL total volume) |
| Product Form | Liquid 2× master mix; requires thawing and thorough mixing before use |
| Quality Control | Each lot verified: >95% correct colonies for 2-fragment control assembly (1 kb vector + 800 bp insert); >80% correct for 4-fragment assembly; no colonies from vector-only control (no insert); sequencing accuracy >98% across assembly junctions; functional testing across DHSalpha, TOP10, and JM109 host strains |
| Key Features | Seamless, scarless cloning without restriction enzymes or ligation; simultaneous assembly of up to 15 fragments; 15-minute assembly time for simple constructs; no sequence constraints on fragment junctions; validated for difficult GC-rich and repetitive sequences; superior to traditional restriction-ligation cloning for multi-fragment assembly |
For research use only, not for clinical use.
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