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| Product Name | T5 Exonuclease, 10 U/µL, for Gibson Assembly and DNA Cloning |
| Catalog No. | IEC-HMM-0072 |
| Description | Recombinant bacteriophage T5 exonuclease (gene D15) that catalyzes 5′→3′ exonucleolytic degradation of linear double-stranded DNA. T5 exonuclease generates long 3′ overhangs used in Gibson Assembly, SLIC cloning, and other homology-based cloning methods. The 5′→3′ directionality ensures asymmetric overhang generation essential for directional cloning of multiple DNA fragments. |
| Intended Use | Gibson Assembly and SLIC (Sequence and Ligation Independent Cloning) for multi-fragment DNA assembly; gene synthesis; site-directed mutagenesis by fragment assembly; cloning vector linearization and processing. |
| Principle / Technology | T5 exonuclease removes nucleotides from 5′ termini of linear dsDNA, creating 3′ single-stranded overhangs; complementary overhangs from adjacent fragments anneal via homology (15-40 bp); in Gibson Assembly, Phusion polymerase fills gaps and Taq ligase seals nicks |
| Detection Method | Gibson Assembly: mix 5′ exonuclease, Phusion polymerase, and Taq DNA ligase with DNA fragments in isothermal buffer; incubate 50 °C for 15-60 minutes; use 2-5 µL assembly reaction for transformation |
| Sample Type | Linear dsDNA fragments with 15-40 bp terminal homology; PCR products, restriction-digested plasmids, synthetic DNA fragments |
| Sensitivity / LOD | Assembles 2-6 DNA fragments in single reaction; total DNA 0.02-0.5 pmol per fragment; assembles fragments 100 bp to >10 kb |
| Reaction Conditions / Protocol | Optimal activity at 37-50 °C in Tris buffer pH 7.5-8.0 with Mg²⁺ (5-10 mM) and DTT (1 mM); 5′-phosphorylated DNA is preferred substrate |
| Components / Formulation | T5 Exonuclease (10 U/µL), 10× T5 Exonuclease Buffer (500 mM Tris-HCl pH 8.0, 50 mM MgCl₂, 10 mM DTT), 100 mM DTT supplement |
| Storage Conditions | -20 °C; enzyme in 50% glycerol — does not freeze solid |
| Shelf Life | 24 months at -20 °C |
| Package Specifications | 1,000 U, 5,000 U, 25,000 U |
| Product Form | Liquid enzyme in 50% glycerol storage buffer |
| Key Features | Core enzyme for Gibson Assembly; efficient 5′→3′ exonuclease activity; creates long 3′ overhangs essential for homology-based cloning; works at 50 °C (compatible with Phusion polymerase temperature range); includes DTT supplement for optimal activity |
| Purity | Recombinant, >99% purity; no detectable 3′→5′ exonuclease, endonuclease, or RNase activity |
| Concentration | 10 U/µL; 1 U = amount of enzyme producing 1 nmol acid-soluble nucleotides from dsDNA in 30 min at 37 °C |
| Activity / Unit Definition | 10,000 U/mL |
| Molecular Weight | As specified per enzyme: Taq ~94 kDa; M-MLV RT ~75 kDa; T4 ligase ~55 kDa; coenzymes as labeled |
| Source / Origin | Recombinant T5 bacteriophage D15 gene product expressed in E. coli |
| pH Range / Optimal pH | pH 7.5–8.5 for polymerase activity; pH 7.5–8.0 for ligation; pH 7.5–8.0 for transcription |
| Shipping Conditions | Cold pack -20 °C |
| Expiration Date / Stability | 24 months at -20 °C; do not heat inactivate — T5 exonuclease is heat-labile and inactivated at 75 °C for 20 min |
| Regulatory / Compliance | For laboratory and research use only; RUO; ISO 9001 |
| Compatibility | Compatible with Gibson Assembly, SLIC, and other homology-based cloning methods; use with Phusion DNA polymerase and Taq DNA ligase for complete Gibson Assembly; DTT is essential for activity — add fresh if buffer is older than 3 months |
| Recommended Buffer System | Tris-HCl, KCl, MgCl₂-based buffers; glycerol-containing storage buffers |
| Application Notes / Precautions | Aliquot enzymes to avoid repeated freeze-thaw; use dedicated nuclease-free workspace; verify enzyme activity for critical applications |
| Batch-to-Batch Consistency | Enzyme specific activity within ±15% of reference lot; functional testing per lot |
For research use only, not for clinical use.
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