T4 Dna Ligase
T4 Dna Ligase - Related Videos
02:57
Assessing the Coexistence of T4 Bacteriophage and E. coli in Murine Fecal Samples
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2026
Source: Pett, N., et al. T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo. J. Vis. Exp. (2024).This video demonstrates the in vivo interaction between T4 bacteriophage and E. coli in the murine intestine, and the fecal spot plating method to quantify their coexistence by measuring bacterial colonies and phage plaques.
09:31
Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
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Cited by 56 •
2016
We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.
12:03
Protocols for C-Brick DNA Standard Assembly Using Cpf1
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Cited by 4 •
2017
CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.
08:56
Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants
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2017
A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.
07:34
DNA Ligation Reactions
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2023
In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an...
FAQs
ATP provides the activation step that prepares a DNA end for ligation. After this activation, T4 DNA ligase can join a 3′ hydroxyl to a neighboring 5′ phosphate, creating the phosphodiester linkage that stabilizes the connection. This ATP dependence distinguishes the enzyme’s reaction from DNA joining processes that do not use ATP in the same way.
Compatible cohesive ends can be joined efficiently because their termini are suitable for direct connection before the phosphodiester bond is sealed. Blunt ends lack that same cohesive compatibility, so their joining requires suitable reaction conditions. This distinction helps researchers select the appropriate DNA-end configuration when designing recombinant DNA assemblies.
The 3′ hydroxyl and neighboring 5′ phosphate provide the specific chemical groups required for forming the DNA backbone connection. T4 DNA ligase seals the nick between these adjacent termini rather than joining arbitrary ends. Preserving this end chemistry is therefore essential when preparing DNA fragments for assembly, cloning, or subsequent analysis.
A typical conceptual workflow brings a DNA fragment and a plasmid vector together so their compatible ends can be connected, then uses T4 DNA ligase to seal the resulting junctions. The product is a recombinant DNA molecule containing the inserted sequence. This approach supports gene insertion into vectors used in molecular bioengineering.
Researchers choose this enzyme when they need stable junctions between DNA fragments, particularly during recombinant DNA assembly or gene insertion into plasmid vectors. It is also useful when engineered DNA molecules must be prepared for cloning and analysis. Its ability to join cohesive ends, and blunt ends under suitable conditions, broadens its practical value.
In bioengineering, the enzyme converts separately prepared DNA pieces into connected constructs that can be examined or used in downstream workflows. Applications include recombinant DNA production, plasmid-based gene insertion, library construction, and preparation of engineered molecules. These uses make ligation a practical connection point between DNA design and experimental cloning or analysis.