T4 Polynucleotide Kinase

Kód produktu: EK0031 Kód výrobce: EK0031 Kód dodavatele: {154DAF51-9AC4-42C5-B560-0FF7CECAEA1F} Výrobce: UAB Fermentas
1 195,48 Kč
988,00 Kč bez DPH
do týdne
500 Units

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

Zobraz detailní popis

Detailní popis

Thermo Scientific 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) (see Reference1).

The enzyme is also a 3'-phosphatase (see Reference2).

Highlights

  • Active in Thermo Scientific restriction enzyme, RT, and T4 DNA Ligase buffers

Applications

  • Labeling of nucleic acids' 5'-termini(see​ Reference3, 4) to be used as (see Figure 1 in Supporting Data):
    • probes for hybridization
    • probes for transcript mapping
    • markers for gel electrophoresis
    • primers for DNA sequencing
    • primers for PCR
  • 5'-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(see​ Reference5, 6)
  • Removal of 3'-phosphate groups(see​ Reference2)

Notes

Polyethylene glycol (PEG) and spermidine improve the rate and efficiency of the phosphorylation reaction (see Reference7). 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(see​ References 1, 2).

10X Reaction Buffer A (for forward reaction) 500mM Tris-HCl (pH7.6 at 25°C), 100mM MgCl2, 50mM DTT, 1mM spermidine
10X Reaction Buffer B (for exchange reaction) 500mM imidazole-HCl (pH6.4 at 25°C), 180mM MgCl2, 50mM DTT, 1mM spermidine and 1mM ADP
24% PEG Solution 24%(w/v) polyethylene glycol 6000
Concentration 10 U/µL
Definition of Activity Unit
  • One unit of the enzyme transfers 1nmol of gamma-phosphate from ATP to 5'-OH DNA in 30min at 37°C.
  • Enzyme activity is assayed in the following mixture: 100mM Tris-HCl (pH8.0), 10mM MgCl2, 5mM DTT, 0.5mM 5'-OH DNA, 0.05mM ATP and 0.1MBq/mL [gamma-33P]-ATP.
Hazardous No
Inactivation Inactivated by heating at 75°C for 10min or by addition of EDTA.
Inhibition Inhibitors: metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 50mM
Molecular Weight The enzyme is a homotetramer. It consists of four identical subunits of 28.9kDa.
Quality Control The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested for labeling 5'-termini of DNA.
Storage Buffer The enzyme is supplied in 20mM Tris-HCl (pH7.5), 25mM KCl, 0.1mM EDTA, 2mM DTT and 50%(v/v) glycerol.
Storage Condition -20 C

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