Ligation of DNA is a critical step in many modern molecular biology workflows. The sealing of nicks between adjacent residues of a single-strand break on a double-strand substrate and the joining of double-strand breaks are enzymatically catalyzed by DNA ligases. The formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate of adjacent DNA residues proceeds in three steps: Initially, the ligase is self-adenylated by reaction with free ATP. Next, the adenyl group is transferred to the 5'-phosphorylated end of the "donor" strand. Lastly, the formation of the phosphodiester bond proceeds after reaction of the adenylated donor end with the adjacent 3' hydroxyl acceptor and the release of AMP. In living organisms, DNA ligases are essential enzymes with critical roles in DNA replication and repair. In the lab, DNA ligation is performed for both cloning and non-cloning applications.
DNA Ligation includes these areas of focus:
Protocols for DNA Ligation
- Double Digest Protocol with Standard Restriction Enzymes
- E. coli DNA Ligase Protocol (M0205)
- Electroporation Protocol (C2989)
- HiFi Taq DNA Ligase (M0647) Protocol
- Ligation Protocol for Cloning with Blunt/TA Ligase Master Mix (M0367)
- Ligation Protocol for Cloning with Instant Sticky-end Ligase Master Mix (M0370)
- Ligation Protocol with T3 DNA Ligase (M0317)
- Ligation Protocol with T4 DNA Ligase (M0202)
- Ligation Protocol with T7 DNA Ligase (M0318)
- NEBNext Quick Ligation Module Protocol (E6056)
- Optimizing Restriction Endonuclease Reactions
- Please see manual (NEB #E7445) for protocols
- Protocol for 9°N DNA Ligase (M0238)
- Protocol for ssDNA/RNA Ligation (M0319)
- Protocol for Taq DNA Ligase (M0208)
- Protocol for the Quick Blunting Kit (E1201)
- Removal of Single-Stranded Extension Protocol using Mung Bean Nuclease (M0250)
- Transformation Protocol (M0367)
- Transformation Protocol (M0370)
Application Notes DNA Ligation
DNA Ligase Brochure
The DNA Ligase brochure provides information about the extensive selection of DNA ligases and ligases master mixes available from NEB.
Molecular Cloning Technical Guide
Download the latest Molecular Cloning Technical Guide for help with product selection, protocols, tips for optimization and trouble-shooting.
Reagents & Tools for Molecular Cloning brochure
Learn about recommended products for cloning in our Reagents and Tools for Molecular Cloning Brochure.
- Properties of DNA and RNA Ligases
- Troubleshooting Guide for Cloning
- Troubleshooting Guide for Ligases
- Troubleshooting Tips for Ligation Reactions
- Tips for Maximizing Ligation Efficiencies
- Traditional Cloning Quick Guide
Other Tools & Resources
High fidelity polymerases are everywhere—but why would you need a high fidelity ligase? And what do we even mean by “fidelity” when we’re talking about ligation? In this webinar, NEB Scientist and ligase expert Greg Lohman discusses mismatch ligation by DNA ligases and the molecular diagnostics applications that depend on the use of high-fidelity DNA ligases like NEB’s HiFi Taq DNA Ligase to detect single base differences in DNA.
Ligation, the process of joining DNA fragments with a DNA ligase, proceeds in three steps. Learn more about ligation with our quick animation.
NEB continues to develop and produce the most extensive commercially available selection of high-quality, and performance-optimized DNA ligases and ligase master mixes for your ligation needs.
The optimal reactant ratio is contingent upon the downstream application.
Polyethylene glycol (PEG) is an important reagent in ligation reactions, find out why.
Find out how the downstream application dictates the best reaction conditions for ligation.
Ligation of blunt ends and single-base overhangs require optimized reaction conditions.