Thermo Scientific™ T4 Polynucleotide Kinase (T4 PNK)
T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5'-OH group of oligonucleotides, ss and dsDNA and RNA.
Manufacturer: Thermo Scientific™ EK0031
T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5'-OH group of single- and double-stranded DNAs and RNAs, oligonucleotides or nucleoside 3'-monophosphates (forward reaction). The reaction is reversible. In the presence of ADP T4 Polynucleotide Kinase exhibits 5'-phosphatase activity and catalyzes the exchange of phosphate groups between 5'-P-oligo-polynucleotides and ATP (exchange reaction).
The enzyme is also a 3'-phosphatase.
- Active in restriction enzyme, RT and T4 DNA Ligase buffers.
- Source: E. coli cells with a cloned pseT gene of bacteriophage T4
- Molecular Weight: The enzyme is a homotetramer. It consists of four identical subunits of 28.9 kDa
- The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested for labeling 5ft.-termini of DNA
- E.coli cells with a cloned pseT gene of bacteriophage T4
- The enzyme is a homotetramer. It consists of four identical subunits of 28.9kDa
- One unit of the enzyme transfers 1nmol of gamma-phosphate from ATP to 5ft.-OH DNA in 30 min. at 37°C
- Enzyme activity is assayed in the following mixture: 100mM Tris-HCl (pH 8.0), 10mM MgCl2, 5mM DTT, 0.5mM 5ft.-OH DNA, 0.05mM ATP and 0.1MBq/ml [gamma-33P]-ATP
- The enzyme is supplied in: 20mM Tris-HCl (pH 7.5), 25mM KCl, 0.1mM EDTA, 2mM DTT and 50% (v/v) glycerol
- 10X Reaction Buffer A (for forward reaction): 500mM Tris-HCl (pH 7.6 at 25°C), 100mM MgCl2, 50mM DTT, 1mM spermidine
- 10X Reaction Buffer B (for exchange reaction): 500mM imidazole-HCl (pH 6.4 at 25°C), 180mM MgCl2, 50mM DTT, 1mM spermidine and 1mM ADP
- 24% PEG Solution: 24% (w/v) polyethylene glycol 6000 Inhibition and Inactivation:
- Inhibitors: metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 50mM
- Inactivated by heating at 75°C for 10 min. or by addition of EDTA
Labeling 5ft.-termini of nucleic acids (3, 4) to be used as probes for hybridization, probes for transcript mapping, markers for gel-electrophoresis, primers for DNA sequencing, primers for PCR; 5ft.-phosphorylation of oligonucleotide, PCR products, other DNA or RNA prior to ligation; Phosphorylation of PCR primers; Detection of DNA modification by the [32P]-postlabeling assay (5, 6); Removal of 3ft.-phosphate groups (2)
Polyethylene glycol (PEG) and spermidine improve the rate and efficiency of the phosphorylation reaction (7). PEG is used in the exchange reaction mixture. As T4 Polynucleotide Kinase is inhibited by ammonium ions, use sodium acetate to precipitate DNA prior to phosphorylation (1, 2).
|metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 50mM|
|The enzyme is a homotetramer. It consists of four identical subunits of 28.9 kDa|
|E. coli cells with a cloned pseT gene of bacteriophage T4|