T4 DNA Ligase

High-Efficiency ATP-Dependent Ligation for Cloning, NGS & Molecular Biology Workflows

Blunt & cohesive

Efficient blunt-end ligation

DNA / RNA hybrids

Seals nicks in double-stranded substrates

No exonuclease

Batch-validated purity

Product overview

T4 DNA Ligase is an ATP-dependent ligase optimized for high-efficiency catalysis of phosphodiester bond formation between the 5′-phosphate terminus of one nucleotide and the 3′-hydroxyl terminus of an adjacent nucleotide in double-stranded DNA (dsDNA), effectively sealing nicks in duplex molecules. It supports repair of single-stranded nicks in double-stranded RNA, DNA/RNA hybrids, and chimeric nucleic acids, but does not catalyze ligation of fully single-stranded polynucleotides. With blunt-end ligation efficiency significantly exceeding that of E. coli DNA ligase, it also supports cohesive-end ligation, DNA/RNA hybrid nick sealing, and gap repair in hybrid duplexes. Rigorous quality control confirms the absence of detectable contaminating exonuclease or nick-translation activities, making it a reliable choice for sensitive molecular workflows.

Features and applications

Features

Broad-Spectrum Ligation Activity: Efficiently seals nicks in dsDNA, dsRNA, DNA/RNA hybrids, and chimeric nucleic acids; explicitly does not ligate fully single-stranded polynucleotides, avoiding off-target artifacts.
Superior Blunt-End Performance: Blunt-termini ligation efficiency is significantly higher than that of wild-type E. coli DNA ligase, solving a common pain point for challenging cloning and NGS library construction workflows.
Ultra-High Purity: Batch-to-batch validation confirms no detectable exonuclease or nick-translation activities, preventing unwanted nucleic acid degradation and ensuring reproducible results.
Workflow Compatibility: Functions optimally with standard ligation buffers and reaction conditions, requiring minimal protocol optimization for most applications.

Applications

NGS Library Preparation: Adapter ligation for amplicon, shotgun, and target enrichment libraries; gap repair in hybrid nucleic acid duplexes
Genetic Cloning: Blunt-end and cohesive-end ligation for vector insertion, gene assembly, and site-directed mutagenesis
Molecular Biology Assays: Ligation-mediated PCR (LM-PCR), linker ligation for ChIP-seq or ATAC-seq, RNA secondary structure probing
RNA-Focused Workflows: Repair of nicks in dsRNA or DNA/RNA hybrids for RNA-seq, Ribo-seq, or in vitro translation assays
Diagnostic R&D: Development of hybridization-based or ligation-dependent molecular detection assays

Components

Component	Specification/Notes
5×Ligase Buffer	5x
T4 DNA Ligase	600 U/ μL

Performance data

High Ligation Efficiency and Specificity

Note: BR3P301 is the catalog number for T4 DNA Ligase. Adapter dimers are a common byproduct of NGS library preparation that reduce usable sequencing data yield; minimal adapter dimer formation improves overall library quality.

Superior Ligation Performance at Low Input

Note: Low-input workflows include single-cell sequencing, cell-free DNA (cfDNA) sequencing, and FFPE-derived library construction where template availability is limited. This performance ensures reliable library construction from precious, limited samples.

Inter-batch Consistency of Ligation

BR3P301 T4 DNA Ligase: Figure 3. Inter-batch consistency of BR3P301 ligation — **Figure 3. Inter-batch consistency of BR3P301 ligation.** Three independent BR3P301 production lots were used to ligate a synthetic dA-tailed substrate. Following 100-fold dilution, qPCR quantification of fully ligated products showed minimal CT value variation across batches, confirming reproducible ligation efficiency and low lot-to-lot variation for robust NGS library construction.

Note: Lot-to-lot consistency is a critical quality metric for clinical and high-throughput NGS workflows, ensuring comparable results across multiple experiments, studies, and production batches.