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.
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 7.8), 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
- 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.
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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. |
