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Specifications:
| Application | Cloning, Transfection | ||
| Storage Temperature | -20°C | ||
| Product Type | Enzymes | Forms | Liquid |
| Product Brand | Thermo Fisher Scientific™ | ||
| Product Grade | Molecular Biology | ||
Thermo Scientific™ XagI (EcoNI) is a high-fidelity restriction enzyme that recognizes the degenerate sequence 5'- CCTNN^NNNAGG -3' / 3'- GGANN^NNNTCC -5' and cleaves within the recognition site. It is widely used in molecular cloning, restriction mapping, genotyping, and Southern blot applications.
XagI (EcoNI) efficiently digests DNA at 37°C in R buffer and is optimized for single, double, and multiplex restriction digestions. It is part of Thermo Scientific’s Five Buffer System, ensuring high specificity, no star activity, and compatibility with downstream applications.
Key Features & Benefits
- Degenerate Sequence Recognition for Versatile Cloning
- Recognizes CCTNN^NNNAGG sequences, providing flexibility in cutting varied DNA targets.
- Ideal for restriction mapping, genetic engineering, and recombinant DNA technology.
- Optimized for Molecular Cloning & Genetic Engineering
- Works best at 37°C in Buffer R, ensuring consistent restriction digestion.
- Part of the Five Buffer System, offering universal enzyme compatibility.
- Fast & Efficient DNA Digestion
- High enzyme activity (10 U/μL) for rapid and reliable restriction digestion.
- Also available as a FastDigest enzyme for faster workflows.
- Compatible with Downstream Applications
- Works seamlessly with DNA ligases, polymerases, and modifying enzymes.
- Produces fragments suitable for recombinant DNA techniques and sequencing.
- Superior Quality & High Purity
- Free from nonspecific nuclease contamination.
- Pre-mixed BSA in reaction buffers enhances enzyme stability.
Product Specifications
| Feature | Details |
|---|---|
| Product Name | Thermo Scientific™ XagI (EcoNI) Restriction Enzyme |
| Recognition Sequence | 5'- CCTNN^NNNAGG -3' / 3'- GGANN^NNNTCC -5' |
| Catalog Number | ER1301 (1,000 units) |
| Enzyme Concentration | 10 U/μL |
| Optimal Temperature | 37°C |
| Compatible Buffer | 10X Buffer R |
| Methylation Sensitivity | Not Sensitive to Dam, Dcm, or CpG Methylation |
| Heat Inactivation | Yes (65°C for 20 minutes) |
| Type IIS Restriction Enzyme | No |
| Shipping Conditions | Ice Packs (International), Dry Ice (Bulk Orders) |
| Storage Conditions | -20°C |
| Unit Size | Each |
Use Cases & Applications
1. Molecular Cloning & Recombinant DNA Technology
- Plasmid & Vector Digestion for Gene Cloning
- Produces sticky ends for directional vector-insert ligation.
- Ensures efficient DNA fragment excision for subcloning applications.
- Double Digestion for Efficient DNA Assembly
- Works well with other Thermo Scientific restriction enzymes.
- Reduces reaction time while maintaining high specificity.
2. Genotyping & Genetic Variation Analysis
- Restriction Fragment Length Polymorphism (RFLP) Analysis
- Identifies genetic variants based on restriction site digestion patterns.
- Used for genotyping, marker-assisted selection, and forensic analysis.
- Single Nucleotide Polymorphism (SNP) Detection
- Helps detect point mutations and insertions/deletions in genomic DNA.
- Supports molecular diagnostics and genetic screening applications.
3. Southern Blotting & DNA Fragment Analysis
- Preparation of DNA Fragments for Hybridization Assays
- Ensures specific and reproducible restriction digestion for probe hybridization.
- Used in gene mapping and epigenetic studies.
- Chromosomal Analysis & Methylation Studies
- Suitable for methylation-dependent restriction analysis.
- Helps in differentiating genetic variations and structural modifications.
4. High-Throughput Cloning & Next-Generation Sequencing (NGS)
- Efficient Restriction Digestion for Library Preparation
- Generates consistent DNA fragments for sequencing library construction.
- Ensures uniform fragment size for high-quality sequencing reads.
- Compatible with Automated Workflows & Liquid Handling Systems
- Ideal for robotic sample processing in high-throughput genomic workflows.
- Eliminates time-consuming buffer exchanges, streamlining DNA digestion workflows.
Recommended Protocol for XagI (EcoNI) Digestion
Standard 50 µL Reaction Setup:
| Component | Volume |
|---|---|
| Plasmid or Genomic DNA | 0.5–1 µg |
| XagI (EcoNI) Restriction Enzyme (10 U/μL) | 1 μL |
| 10X Buffer R | 5 μL |
| Nuclease-Free Water | Adjust to 50 μL |
Incubation & Post-Digestion Processing
- Digest at 37°C for 1 hour for complete restriction.
- Heat inactivate at 65°C for 20 minutes, if necessary.
- Analyze digestion products on a 1–2% agarose gel using ethidium bromide or SYBR Safe staining.
Storage & Handling Instructions
- Storage Conditions:
- Store at -20°C for long-term enzyme stability.
- Avoid freeze-thaw cycles to maintain enzyme activity.
- Handling Precautions:
- Use sterile, nuclease-free reagents and pipette tips to prevent contamination.
- Work in a DNA-free environment to ensure reproducibility.
- Disposal Guidelines:
- Dispose of enzyme and buffers according to biosafety waste regulations.
- Use approved chemical waste disposal protocols for reaction waste.
Why Choose Thermo Scientific™ XagI (EcoNI)?
✅ Recognizes degenerate sequence CCTNN^NNNAGG for flexible cloning
✅ Optimized for molecular cloning, restriction mapping, and genetic engineering
✅ Compatible with the universal Tango buffer for double digestions
✅ Reliable and consistent performance in traditional and high-throughput workflows
✅ Methylation-insensitive enzyme for robust DNA digestion
Thermo Scientific™ XagI (EcoNI) is a highly precise, fast-acting restriction enzyme, making it a trusted choice for molecular biology, cloning, and DNA analysis applications.