T4 Polynucleotide Kinase (PNK) catalyses the transfer of the g-phosphate from ATP to the 5'-hydroxyl of single- or double-stranded DNA, RNA, and nucleoside 3'-monophosphates. The enzyme also removes the 3'-phosphate from 3'-phosphoryl polynucleotides, deoxyribonucleoside 3'-monophosphates, and deoxyribonucleoside 3', 5'-diphosphates to form a 3'-hydroxyl group.
Unit Definition
Under standard conditions, one unit converts 1nmole of 32P from [g-32P]-ATP into an acid-insoluble form in 30 minutes at 37°C in 33 mM Tris-acetate, pH 7.8, 66 mM potassium acetate, 10mM magnesium acetate, 0.5mM DTT, 1.0mM ATP, and 0.1mg/ml micrococcal nuclease-treated calf thymus DNA as substrate.
Storage Buffer
50% glycerol containing 50mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.1mM EDTA, 0.1% Triton® X-100, and 1 mM DTT.
T4 PNK 10X Reaction Buffer
330mM Tris-acetate, pH 7.8, 660mM potassium acetate, 100mM magnesium acetate, and 5 mM DTT. A 10mM solution of ATP for non-isotopic applications is available separately.
Quality Control
T4 PNK is tested in 5'-phosphorylation of nucleic acids and is free of detectable nuclease activities.
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