Polynucleotide Kinase T4

T4 polynucleotide kinase catalyzes the transfer of the terminal phosphate of ATP (gamma-orthophosphate) to the 5'-hydroxyl termini of ribo- and deoxyribonucleotides. In the presence of ADP the enzyme also catalyzes an exchange reaction. In the exchange reaction, an excess ADP causes the enzyme to transfer the 5' terminal phosphate from phosphorylated DNA to ADP. The DNA thus dephosphorylated is then rephosphorylated by transfer of radiolabeled gamma-phosphate from gamma 32P ATP.

Applications:
•5’ Labeling of nucleic acids
- probes for hybridization
- probes for transcript mapping
- markers for gel electrophoresis
- primers for DNA sequencing
- primers for PCR
•5’phosphorylation of oligonucleotides, PCR products, other DNA or RNA prior to ligation
•Phosphorylation of PCR primers
•Detection of DNA modification by the [32P]-postlabeling assay
•Removal of 3’-phosphate groups

Source:
E. coli infected with T4 phage

Form:
Supplied as a liquid in 20mM Tris-HCl, pH 7.5, 25mM KCl, 0.1mM EDTA, 2mM DTT, 50% glycerol,.

Concentration:
10u/ul

Unit Definition:
One unit will incorporate 1 nanomole of gamma phosphate from ATP to 5’-OH DNA in 30 minutes at 37ºC.

Activity Assay Buffer:
100mM Tris-HCl, pH 8.0, 10mM MgCl2, 5mM DTT, 0.5mM 5’-OH DNA, 0.05mM ATP, 0.1MBq/ml [g33p]-ATP.

Inhibitors:
Metal chelators, phosphate and ammonium ions, KCl and sodium chloride at a concentration higher than 50mM.

Inactivation:
Heat at 75ºC for 10 min or by the addition of EDTA.

Supplied with:
P4410A: Forward Reaction Buffer (10X):
Supplied as a liquid in 500mM Tris-HCl, pH 7.6,n100mM MgCl2, 50mM DTT, 1mM Spermidine.

P4410B;, Exchange Reaction Buffer (10X):
Supplied as a liquid in 500mM Imidazole-HCl, pH 6.4, 180mM MgCl2, 50mM DTT, 1mM Spermidine, 1mM ADP.

P4410C: PEG Solution:
Supplied as a liquid, 24% w/v PEG 6000.

Quality Control:
1. Endodeoxyribonuclease Assay: No detectable degradation was observed after incubation of supercoiled plasmid DNA with T4 Polynucleotide Kinase.

2. Ribonuclease Assay: No detectable degradation was observed after incubation of [3H] with T4 Polynucleotide Kinase.

Storage and Stability:
May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for 6 months after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.