Applications include end-labeling and insert preparation for cloning.
T4 Polynucleotide Kinase catalyzes the transfer and exchange of gamma-phosphate of ATP to the 5´-hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3´-monophosphates.
Source: Purified from a strain of E. coli that carries a T4 Polynucleotide Kinase expression plasmid.
Applications:
End-labeling DNA for probes and DNA sequencing (1)
Addition of 5´-phosphates to oligonucleotides to allow subsequent ligation (2)
Heat inactivation: 70°C for 15 minutes.
Storage: Enzyme and buffer should be stored at -20°C. Enzyme is stable for 12 months if handled properly.
Additional Information:
Ammonium ions are potent inhibitors of T4 Polynucleotide Kinase. DNA should not be precipitated from or dissolved in buffers containing ammonium salts.
Protruding 5’-overhangs are more efficiently phosphorylated than blunt- or recessed 5’ overhangs.
References:
Sambrook, J. and Russell, W. (2001) Molecular Cloning: A Laboratory Manual, (3rd ed.), 9.55-9.56, 9.68-9.69.
Sambrook, J. and Russell, W. (2001) Molecular Cloning: A Laboratory Manual, (3rd ed.), 13.16-13.17.