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Cat. No.: T4DLF-1k (for 1KU)

Cat. No.: T4DLF-5k (for 5KU)

Description

T4 DNA Ligase (Fast) is produced by Escherichia coli carrying the T4 bacteriophage gene 30. This enzyme catalyzes the formation of a phosphodiester bond between the 5'-phosphate group and the 3'-hydroxyl group of double-stranded DNA or RNA. It can repair single-strand breaks in double-stranded DNA, RNA, or DNA/RNA hybrids, and can ligate DNA fragments with either sticky or blunt ends. However, it is inactive towards single-stranded nucleic acids. It is primarily used for cloning of restriction endonuclease-digested DNA fragments, site-directed mutagenesis, cloning of PCR products, linear DNA self-ligation, and repair of double-strand DNA breaks. T4 DNA Ligase (Fast) requires ATP as a cofactor and can complete sticky end ligation reactions at room temperature in just 10 minutes.

Unit Definition

Under 37°C conditions, 1 Weiss unit of enzyme catalyzes the conversion of 1 nmol of [32PPi] to an adsorbed state on activated charcoal within 20 minutes. 1 Weiss unit is equivalent to approximately 200 cohesive end ligation units (CEU), which is comparable to ligating 50% of HindIII-digested λDNA fragments within 30 minutes at 16°C.

Enzyme Activity Assay Conditions 

Enzyme activity is tested in the following reaction mixture: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl₂, 66 μM ATP, 10 mM DTT, and 3.3 μM [³²PPi].

Quality Control

• Residual Endonuclease Activity Detection: Under 37°C conditions, incubate 200 U of T4 DNA Ligase (Fast) with 1 μg of pUC19 DNA for 4 hours. No conversion of covalently closed circular DNA to DNA with nicks was detected.
• Residual Exonuclease Activity Detection: Incubate the enzyme solution with double-stranded DNA substrate at 37°C for 16 hours. Analyze by DNA electrophoresis to confirm no change in the double-stranded DNA substrate.
• Blue-White Screening: Under room temperature conditions, incubate pUC57 DNA/HindIII, pUC57 DNA/PstI, or pUC57 DNA/SmaI digested products with 30 U of T4 DNA Ligase (Fast) for 1 hour. Subsequently, transform the ligation products into E. coli XL1-Blue competent cells. Detection reveals fewer than 1% white colonies.

Storage

Store at -20°C.

DNA Ligase Products:

• T4 DNA Ligase
• Rapid T4 DNA Ligase
• T4 DNA Ligase (Fast)
• T7 DNA Ligase
• E. coli DNA Ligase
• PBCV-1 DNA Ligase
• T3 DNA Ligase
• Taq DNA Ligase
• Pfu DNA Ligase
• High-Fidelity DNA Ligase
• High-Salt DNA Ligase

SBS Genetech is recognized as one of the global major leading industry players in Ligase Market by third-party market researchers. For more details, please visit Ligase Market - A Global and Regional Analysis Focus on Product, Source, Application, End User, and Country - Analysis and Forecast, 2022-2032

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