T4 Polynucleotide Kinase, Cloned, 3,000 U

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.

Applications

• 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.

References

1. Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York.
2. 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.