Thermo Scientific™ T4 DNA Ligase (5 U/μL)

Thermo Scientific™ T4 DNA Ligase is a high-performance enzyme that catalyzes the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini in DNA or RNA. The enzyme plays a crucial role in repairing single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids, and is widely used in molecular biology applications, particularly in cloning, site-directed mutagenesis, and ligation of DNA fragments.

T4 DNA Ligase is particularly effective for joining DNA fragments with either cohesive or blunt ends, making it an essential tool in a variety of research applications. The enzyme does not act on single-stranded nucleic acids, ensuring specificity for duplex DNA or RNA.

T4 DNA Ligase requires ATP as a cofactor for its enzymatic activity and is optimized for use in common buffers such as Thermo Scientific restriction enzyme, PCR, and RT buffers (when supplemented with ATP).

Key Features:

• Rapid Action: Sticky-end ligation can be completed in as little as 10 minutes at room temperature, ensuring efficiency in experimental setups.
• Efficient Blunt-End Ligation: Supplied with PEG solution, which enhances blunt-end ligation, making it easier to join blunt-ended DNA fragments.
• Versatility: Active in various buffers, including Thermo Scientific restriction enzyme, PCR, and RT buffers, making it adaptable for a wide range of molecular biology applications.

Applications:

1. Cloning of Restriction Enzyme Generated DNA Fragments:
   • Used for ligating DNA fragments produced by restriction enzymes, essential for cloning applications.
2. Cloning of PCR Products:
   • Enables the ligation of PCR-generated DNA fragments into vectors for cloning or expression studies.
3. Joining of Double-Stranded Oligonucleotide Linkers or Adaptors:
   • Ideal for attaching oligonucleotide linkers or adaptors to DNA molecules in various genetic manipulation protocols.
4. Site-Directed Mutagenesis:
   • Essential for mutagenesis experiments, enabling the introduction of specific mutations into DNA sequences.
5. Amplified Fragment Length Polymorphism (AFLP):
   • Used in AFLP analysis, which is a powerful tool for identifying genetic markers.
6. Ligase-Mediated RNA Detection:
   • Employed in RNA detection methods to join RNA fragments or detect specific RNA sequences in gene expression studies.
7. Nick Repair in Duplex DNA, RNA, or DNA/RNA Hybrids:
   • T4 DNA Ligase repairs single-strand nicks in duplex DNA and RNA, or DNA/RNA hybrids, to maintain the integrity of nucleic acids.
8. Self-Circularization of Linear DNA:
   • Can facilitate the self-circularization of linear DNA, a critical step in certain types of DNA cloning.

Contents:

• T4 DNA Ligase
• 10X T4 DNA Ligase Buffer
• 50% PEG Solution (for efficient blunt-end ligation)

Important Notes:

• When preparing for agarose gel electrophoresis, it is recommended to incubate samples with 6X DNA Loading Dye & SDS Solution at 70°C for 5 minutes or 65°C for 10 minutes, then chill on ice to prevent band shifting caused by T4 DNA Ligase binding to DNA.
• In transformation protocols, the volume of the ligation reaction should not exceed 10% of the competent cell volume.
• Precipitation of the ligation mixture is recommended before electro-transformation to remove T4 DNA Ligase using spin columns or chloroform extraction.

Specifications:

Storage:

• Store at ambient temperature for regular use. Ensure proper handling to maintain enzyme activity and shelf life.

The Thermo Scientific™ T4 DNA Ligase (5 U/μL) is an essential enzyme for a variety of applications in molecular biology, from cloning and site-directed mutagenesis to DNA repair and ligase-mediated RNA detection. Its versatility, fast action, and ability to handle both sticky and blunt-ended DNA fragments make it a crucial reagent for researchers conducting genetic manipulation and other nucleic acid-based experiments.