Thermo Scientific™ NotI (10 U/μL)

Thermo Scientific™ NotI is a high-fidelity restriction enzyme that specifically recognizes and cleaves the palindromic DNA sequence 5'- GC↓GGCCGC -3' / 3'- CGCCGG↑CG -5'. This enzyme is widely used in molecular cloning, genetic engineering, restriction mapping, and Southern blot analysis.

NotI performs efficient and precise DNA cleavage at 37°C in Buffer O, making it ideal for single and double restriction digestion experiments. It is CpG methylation-sensitive, ensuring selective cleavage based on DNA methylation status.

Key Features & Benefits

1. High-Specificity Restriction Digestion
   • Recognizes GC^GGCCGC sequences with precise cleavage.
   • Generates sticky ends for efficient ligation and cloning.
2. Optimized for Molecular Cloning & Genetic Engineering
   • Works best at 37°C in Buffer O, ensuring consistent DNA cleavage.
   • Part of the Thermo Scientific Five Buffer System, allowing for universal enzyme compatibility.
3. Fast & Efficient DNA Digestion
   • High enzyme activity (10 U/μL) for rapid DNA processing.
   • Also available in FastDigest format for accelerated reactions.
4. Methylation Sensitivity for Epigenetic Studies
   • Sensitive to CpG methylation, making it useful for methylation-dependent restriction analysis.
   • Not sensitive to Dam or Dcm methylation, ensuring compatibility with many plasmid and genomic DNA sources.
5. Superior Quality & High Purity
   • Free from nonspecific nuclease contamination.
   • Pre-mixed BSA in reaction buffers enhances enzyme stability.

Product Specifications

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 Seamless DNA Assembly
  • Works well with other restriction enzymes, enabling efficient multi-step cloning.
  • Reduces reaction time with high restriction site specificity.

2. Genotyping & Genetic Variation Analysis

• Restriction Fragment Length Polymorphism (RFLP) Analysis
  • Identifies genetic variants based on restriction enzyme digestion patterns.
  • Used in genotyping, population genetics, and forensic analysis.
• Single Nucleotide Polymorphism (SNP) Detection
  • Helps identify 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
  • Detects methylation status in CpG-sensitive restriction sites.
  • Useful for epigenetic profiling and structural variation studies.

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 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 NotI Digestion

Standard 50 µL Reaction Setup:

Incubation & Post-Digestion Processing

1. Digest at 37°C for 1 hour for complete restriction.
2. Heat inactivate at 80°C for 20 minutes, if necessary.
3. 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™ NotI?

✅ High specificity for GC^GGCCGC restriction sites with no star activity

✅ 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

✅ CpG methylation-sensitive enzyme for epigenetics and genotyping studies

Thermo Scientific™ NotI is a highly precise, fast-acting restriction enzyme, making it a trusted choice for molecular biology, cloning, and DNA analysis applications.