Thermo Scientific™ FastDigest Eco31I (IIs class)

Thermo Scientific™ FastDigest Eco31I (IIs Class) is a high-fidelity Type IIS restriction enzyme that recognizes the asymmetric sequence 5'- GGTCTC(N1/5)↓ -3' / 3'- CCAGAG(N5)↑ -5' and cleaves outside its recognition site, generating 4-base overhangs. This property makes it particularly useful for Golden Gate Assembly, scarless cloning, synthetic biology, and DNA engineering applications.

FastDigest Eco31I (BsaI) is optimized to rapidly cleave DNA within 5–15 minutes at 37°C using the universal FastDigest Buffer system, allowing seamless single, double, and multi-enzyme digestion without the need for buffer changes or additional cleanup steps. It is fully compatible with other FastDigest enzymes and works efficiently with downstream DNA ligation and modification enzymes.

Key Features & Benefits

1. Type IIS Restriction Enzyme for Precision Cloning
   • Recognizes GGTCTC(N1/5)↓ sites and cleaves outside its recognition sequence, generating customizable 4-base overhangs.
   • Ideal for Golden Gate Assembly, synthetic biology, and scarless cloning.
2. Ultra-Fast DNA Digestion (5–15 Minutes)
   • Enables high-efficiency digestion in a single step, reducing reaction time.
   • Works seamlessly in FastDigest and FastDigest Green buffers.
3. No Star Activity for High-Fidelity Digestion
   • Ensures precise restriction digestion with no off-target cuts.
   • Maintains DNA sequence integrity for downstream applications.
4. Direct Gel Loading with FastDigest Green Buffer
   • Allows immediate electrophoresis without additional sample preparation.
   • Includes tracking dyes and density reagents for streamlined workflow.
5. Compatible with DNA Modifying Enzymes for Seamless Cloning
   • Supports direct addition of T4 DNA Ligase, Klenow Fragment, Alkaline Phosphatase, and T4 DNA Polymerase.
   • Ensures efficient transition from restriction digestion to ligation and cloning.

Product Specifications

Use Cases & Applications

1. Molecular Cloning & Recombinant DNA Technology

• Golden Gate Assembly for Modular Cloning
  • Produces customizable overhangs for seamless DNA fragment assembly.
  • Used in synthetic biology and restriction-free cloning workflows.
• Scarless DNA Assembly for Synthetic Biology
  • Enables restriction-site-free DNA fragment ligation.
  • Supports ligation-independent cloning and multi-fragment assemblies.

2. Genotyping & Genetic Variation Analysis

• Restriction Fragment Length Polymorphism (RFLP) Analysis
  • Identifies genetic variations based on restriction site digestion.
  • Used in genotyping, forensic analysis, and population genetics research.
• 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 accurate and reproducible restriction digestion for Southern blot analysis.
  • Useful in genomic mapping and methylation profiling.
• Chromosomal Analysis & Methylation Studies
  • Detects methylation-sensitive restriction sites in genomic DNA.
  • Used for epigenetics and imprinting research.

4. High-Throughput Cloning & Next-Generation Sequencing (NGS)

• Efficient Restriction Digestion for DNA Library Preparation
  • Generates consistent DNA fragments for sequencing library construction.
  • Ensures uniform fragment size distribution 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 FastDigest Eco31I (BsaI) Digestion

Standard 50 µL Reaction Setup:

Incubation & Post-Digestion Processing

1. Digest at 37°C for 5–15 minutes for complete restriction.
2. Heat inactivate at 65°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™ FastDigest Eco31I (IIs Class)?

✅ Type IIS restriction enzyme with flexible overhangs for modular DNA assembly

✅ Optimized for Golden Gate Assembly & synthetic biology applications

✅ Compatible with universal FastDigest Buffers for rapid digestion

✅ Efficient 5–15 minute digestion for high-throughput workflows

✅ Precise and reproducible cleavage for molecular cloning and gene editing

Thermo Scientific™ FastDigest Eco31I (BsaI) is a high-precision, fast-acting restriction enzyme, making it a trusted choice for molecular biology, synthetic biology, and DNA engineering applications.