Digested DNA typically possesses a 5′ phosphate group that is required for ligation. In order to prevent self-ligation, the 5′ phosphate can be removed prior to ligation. Dephosphorylation of the 5′ end prohibits self-ligation, enabling the researcher to manipulate the DNA as desired before re-ligating. Generally, it is a good idea to dephosphorylate the linearized vector to facilitate ligation of recombinant DNA molecules. In fact, vector self-ligation increases the background activity of the cloning process. Dephosphorylation can be accomplished using any of a number of phosphatases, including the Quick Dephosphorylation Kit (NEB #M0508), Shrimp Alkaline Phosphatase (rSAP) (NEB #M0371), Calf Intestinal Alkaline Phosphatase (CIP) (NEB #M0290) and Antarctic Phosphatase (NEB #M0289).
Protocols for DNA Dephosphorylation
- Affinity Purification and On-column Cleavage (E6901)
- Construction of the Fusion Plasmid (E6901)
- Enzymatic PCR Cleanup Protocol
- Fusion Constructs (E6901)
- Fusion Protein Expression (E6901)
- M13mp18 Phage Transfection
- Preparation of Media and Solutions (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Protocol for Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289)
- Protocol for Dephosphorylation of 5´-ends of DNA using Quick Dephosphorlyation Kit (M0508)
- Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)
- Protocol for Dephosphorylation of 5´-ends of DNA using CIP (NEB #M0290)
- Simplified Expression and Purification Protocol (E6901)
Common Applications of Exonucleases and Endonucleases
Properties of Exonucleases and Endonucleases
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Dephosphorylation is the process by which phosphate groups are removed from a molecule by a phosphatase. Removal of phosphate groups from a DNA fragment can prevent ligation. Learn more about dephosphorylation and phosphatases.