Single- or double-stranded DNA with a 5′-hydroxyl terminus has to be phosphorylated prior to ligation as 5′ phosphate groups are required for ligation. Phosporylation of DNA ends with phosphate radioisotopes can also be used to label DNA in preparation of DNA for subsequent detection, isolation and sequencing applications. A number of polynucleotide kinases, including T4 PNK (NEB #M0201) and T4 PNK (3' phosphatase minus) (NEB #M0236), can be used to transfer the γ-phosphate of ATP to a 5′ terminus of DNA.
Protocols for DNA Phosphorylation
- Affinity Purification and On-column Cleavage (E6901)
- Construction of the Fusion Plasmid (E6901)
- Fusion Constructs (E6901)
- Fusion Protein Expression (E6901)
- Non-radioactive Phosphorylation with T4 PNK or T4 PNK (3´ phosphatase minus)
- Preparation of Media and Solutions (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Protocol for the Quick Blunting Kit (E1201)
- Radioactive Labeling with T4 PNK or T4 PNK (3´ phosphatase minus)
- Simplified Expression and Purification Protocol (E6901)
Common Applications of Exonucleases and Endonucleases
Properties of Exonucleases and Endonucleases
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Phosphorylation is the process by which phosphate groups are added to a molecule by a kinase. The phosphorylation status of a fragment of DNA can influence its ability to proceed in reactions. Learn more about phosphorylation and kinases.