Thermo Scientific™ FastDigest™ FokI, 100 μL

Thermo Scientific™ FastDigest FokI Restriction Enzyme

The Thermo Scientific™ FastDigest FokI is a Type IIS restriction enzyme that recognizes the sequence GGATG(N9/N13)^ and efficiently cleaves DNA at specific positions downstream of the recognition site. This enzyme is part of the advanced FastDigest™ enzyme line, designed to provide fast, reliable, and convenient DNA digestion. It is fully active in universal FastDigest Buffers, allowing compatibility with downstream applications.

Recognition Site and Cleavage Pattern

• Recognition Sequence:
  • 5' G G A T G N₉↓
  • 3' C C T A C N₁₃↑
• Cleavage Details:
  • The enzyme cleaves DNA 9 bases downstream on the top strand and 13 bases downstream on the bottom strand, producing 5' protruding ends.
• Isoschizomers: Recognized by other enzymes such as BstF5I and BtsCI.

Buffer Compatibility and Reaction Conditions

1. Universal Buffer System:
   • Works with 10X FastDigest Buffer (colorless) and 10X FastDigest Green Buffer, supporting DNA modification enzymes such as:
     • Klenow Fragment
     • T4 DNA Ligase
     • Alkaline Phosphatase
     • T4 DNA Polymerase
   • Enables one-step reactions without the need for intermediate buffer changes.
2. Direct Gel Loading:
   • The Green Buffer contains tracking dyes and a density reagent, making digestion products ready for electrophoresis directly after incubation.
3. Short Incubation Time:
   • DNA digestion is complete within 5–15 minutes at 37°C, enhancing workflow efficiency.
4. Heat Inactivation:
   • Heat inactivation can be performed at 65°C to terminate enzyme activity when required for subsequent applications.
5. No Star Activity:
   • Optimized reaction conditions prevent nonspecific cleavage, ensuring high specificity and reliable results.

Key Features

1. Fast and Efficient Digestion:
   • Rapid processing (5–15 minutes) minimizes experimental time while maintaining high specificity.
2. Universal Buffer Compatibility:
   • All FastDigest enzymes operate with the same buffer, simplifying experimental workflows.
3. Downstream Integration:
   • Enzymes for DNA modification or ligation can be directly added to the reaction mix without requiring DNA purification steps.
4. Broad Application Range:
   • Suitable for molecular cloning, genotyping, and other restriction-based techniques.
5. Heat-Sensitive Inactivation:
   • Allows control over enzyme activity, ensuring compatibility with multi-step protocols.

Applications

The FastDigest FokI enzyme is well-suited for the following molecular biology techniques:

• Molecular Cloning: Precise cleavage of DNA to facilitate recombinant construct creation.
• Restriction Site Mapping: Identifying and characterizing restriction sites in DNA.
• Southern Blot Analysis: Detection of specific DNA sequences using restriction digestion.
• Restriction Fragment Length Polymorphism (RFLP): Genetic variation analysis through restriction patterns.
• SNP Analysis: Studying single nucleotide polymorphisms for genotyping.
• DNA Fragment Preparation: Generating fragments for sequencing or further processing.

Specifications

• Recognition Site: GGATG(N9/N13)^.
• Methylation Sensitivity:
  • Not sensitive to Dam, Dcm, or CpG methylation.
• Optimal Temperature: 37°C.
• Cleavage Type: Produces 5' protruding ends.
• Heat Inactivation: 65°C.
• Type IIS Restriction Enzyme: Cleaves outside the recognition site.
• Quantity: 100 μL (100 reactions).
• Buffer Compatibility: Universal FastDigest Buffer and FastDigest Green Buffer.
• Storage: Store at –20°C.

Contents and Storage

Each package contains:

• FastDigest FokI enzyme (100 μL, 100 reactions).
• 10X FastDigest Buffer (1.0 mL).
• 10X FastDigest Green Buffer (1.0 mL).

Store all components at –20°C to maintain stability and enzymatic activity.

Notes and Considerations

1. Green Buffer vs. Colorless Buffer:
   • The Green Buffer simplifies electrophoresis workflows by including tracking dyes and a density reagent.
   • Use the colorless buffer for fluorescence-based analyses (e.g., UV concentration measurements).
2. Type IIS Restriction Enzyme:
   • Unlike typical restriction enzymes, FokI cleaves DNA at a specific distance from its recognition site, making it ideal for applications requiring precise fragment generation.
3. Compatibility with Downstream Applications:
   • DNA modifying enzymes retain full activity in the FastDigest Buffer, ensuring seamless transitions to ligation, polymerase reactions, or other downstream processes.
4. Research Use Only:
   • Not intended for diagnostic procedures.

The Thermo Scientific™ FastDigest FokI enzyme combines precision, speed, and convenience, making it a reliable tool for advanced molecular biology workflows. Its universal buffer compatibility, absence of star activity, and optimized reaction conditions make it suitable for a variety of restriction-based applications, enhancing productivity in research labs.