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T4 Polynucleotide Kinase

Features: End-labeling DNA or RNA, addition of 5’ -phosphates to oligonucleotides, removal of 3’ -phosphoryl groups.
T4 Polynucleotide Kinase
Description: Catalyses transfer and exchange of Pi from g position of ATP to 5’-hydroxyl terminus of polynucleotides (double and single-stranded DNA and RNA) and nucleoside 3’-monophosphates. Polynucleotide kinase also catalyses the removal of 3’-phosphoryl groups from 3’-phosphoryl polynucleotides and  deoxynucleoside 3’-diphosphates (1).
Purified from E.coli strain carrying T4 polynucleotide kinase overproducing plasmid.
Unit definition: 1 unit of enzyme transfers 1 nanomole of g-phosphate from ATP to 5’-OH DNA in 30 min at 37o C.
Activity assay: 100 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM DTT, 0.5 mM 5’-OH-DNA, 66 mM [g-32P] ATP (5 x 106 cpm/ mmole, 0.26 mM hydroxyl-terminated calf thymus DNA (1).
Storage conditions: –20O C in 50 mM KCl, 10 mM Tris-HCl (pH 7.6), 0.1 mM EDTA, 1 mM DTT, 0.1 mM ATP and 50% glycerol.
Quality control: Endo-, exodeoxyribonucleases, ribonucleases, phosphatase free.
  1.  DNA or RNA 5'-end labelling (2).
  2. Incorporation of  phosphates in 5'-hydroxyle of oligonucleotides to allow subsequent ligation.
  3. Removal of 3’-phosphoryl group.