Phosphorylate 5' hydroxy ends of ssDNA and dsDNA, RNA, and nucleoside 3´ monophosphates
- Robust: Highly active enzyme that readily phosphorylates 5' hydroxl ends of nucleic acids
- Flexible: Use newly phosphorylated nucleic acids in a variety of applications such as cloning, next gen sequencing library prep, and preparation of labeled nucleic acids
T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the γ-phosphate from ATP to the 5´ hydroxyl of ssDNA and dsDNA, 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.
- Labeling of 5´ termini of DNA and RNA with 32P or 33P for DNA sequencing, blot-hybridization experiments, or transcript mapping using Mung Bean Nuclease, S1 nuclease, or other nucleases.1,2
- Phosphorylation of oligonucleotide linkers and other DNA or RNA molecules prior to ligation, or for use in ligation amplification reactions with Ampligase® Thermostable DNA Ligase.
- Preparation of labeled DNA or RNA molecular weight markers for gel electrophoresis and chromatography.
Unit Definition: One unit of T4 Polynucleotide Kinase converts 1 nmol of 32P from [γ-32P]-ATP into an acid-insoluble form in 30 minutes at 37°C under standard assay conditions.
Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 0.1% Triton® X-100, and 1 mM DTT.
T4 PNK 10X Reaction Buffer: 330 mM Tris-acetate (pH 7.5), 660 mM potassium acetate, 100 mM magnesium acetate, and 5 mM DTT. A 10 mM solution of ATP for nonisotopic applications is available separately.
Quality Control: T4 PNK is tested in 5´ phosphorylation of nucleic acids and is free of detectable exo- and endonuclease and RNase activities.
- Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York.
- Chaconas, G. et al. (1980) Meth. Enzymol. 65, 75.
Figure 1. The absence of DNA exonuclease activity in Epicentre's T4 Polynucleotide Kinase. 40-mer oligonucleotides with (5´-PO4) or without (5´-OH) phosphorylated 5´-termini were incubated with the indicated number of units of T4 PNK in 1X T4 PNK Reaction Buffer for 16 hours at 37°C. Electrophoresis of the incubation mixtures in a 15% polyacrylamide/8 M urea gel demonstrates no degradation of the oligonucleotides.