T4 Polynucleotide Kinase

Polynucleotide kinase, which catalyzes the phosphorylation of 5' hydroxyl termini of double- and single-straned DNA or RNA.

Descripiton:

• Catalyzes the transfer of the g-phosphate of ATP to a 5'-OH terminus in DNA or RNA.
• Contains 3'-phosphatase activity (1).
• Used for 5'-end labeling of nucleic acids prior to DNA or RNA sequencing (2, 3).
• Phosphorylates synthetic linkers and fragments of DNA or RNA prior to ligation.
• Labels 5'-termini prior to partial restriction enzyme digestion.

  Unit Definition: One unit is defined as the amount of enzyme required to transfer 1 nmol of g-phosphate from ATP to the 5'-OH termini of salmon sperm DNA fragments in 30 min at 37°C (1).

  Storage Conditions: store at -20°C.

  Storage Buffer: 50 mM Tris-HCl (pH 7.6 at 22°C), 25 mM KCl, 1 mM dithiothreitol, 0.1 M ATP, 0.1 mM EDTA and 50% (v/v) glycerol.

  Assay Conditions: 70 mM Tris-HCl (pH 7.6 at 22°C), 10 mM MgCl2, 5 mM dithiothreitol, 27 nmol of DNA-phosphorus (5'-OH terminated salmon sperm DNA) and 70 nM [ α-³²P]ATP. Reaction volume is 100 µl.

  Quality Control: All preparations are tested for contaminating endonuclease, exonuclease and nonspecific RNase activities, along with functional testing in end-labeling reactions.

  References:
  1.Richardson, C.C. (1971) Progress in Nucleic Acids Research and Molecular Biology 2, 815-828.2. Maxam, A. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560-56.
  3. Donis-Keller, H. (1980) Nucleic Acids Res. 8, 3133-3142.