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Cat. No.: T4PK-200 (for 250U)

Cat. No.: T4PK-2000 (for 2500U)

Description

T4 Polynucleotide Kinase can catalyze the γ position phosphate group of ATP to the 5' - hydroxy end or of the oligonucleotide strands (double-stranded or single-stranded DNA or RNA) and the 3' - monophosphate nucleoside. T4 polynucleotide kinase also has 3' phosphatase activity, which hydrolyzes 3' - phosphate group from 3' phosphate terminal of the oligonucleotide, deoxyribonucleoside 3' - monophosphate and deoxyribonucleoside 3 '- diphosphate. With modification, the 3' phosphatase activity of T4 Polynucleotide Kinase is lost, but all kinase activity remains.

Unit Definition

One unit is defined as the amount of enzyme required to catalyze the incorporation of 1nmol acid insoluble [³²P] within 30 minutes at 37°C.

Components

T4 Polynucleotide Kinase (10 U/μl):    20 μl/200 μl
10×T4 PNK Buffer:    1 ml/1 ml×2
10mM ATP:    100 μl/500 μl

Applications

• Phosphorylate the 5' end of DNA or RNA for ligation reaction.

• Label the 5' end of the oligonucleotide with a phosphate group at the 3' end.

• Phosphorylate the phosphorylated 5' single nucleotide at the 3' end to prepare pNp substrate for addition to the 3' end of DNA or RNA

• Mark the terminal of DNA or RNA to use as a probe and DNA sequencing

Heat Inactivation

65°C for 20min

Storage

The minimum shelf life is 3 years at -20°C.

Note

• 1X T4 PNK Buffer: 70 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 5 mM DTT, 37°C incubation.

• In radiolabeling experiments, 1 × T4 polynucleotide kinase reaction buffer, 50 pmol γ- [³²P] ATP, and 20 units of the enzyme are incubated at 37°C for 30 minutes.

• T4 polynucleotide kinase requires ATP to perform its activity, but in order to be suitable for high-activity radiolabeling reactions, the reaction buffer provided with the enzyme does not contain ATP. Therefore, the final concentration of 0.5~1mM ATP should be added separately when phosphorylating the nucleic acid.

• To improve the phosphorylation efficiency of the blunt end or the 5' depressing end, the DNA solution can be heated at 70°C for 5 minutes, then cooled on ice, and 5% (W/V) PEG-8000 can be added before adding T4 polynucleotide kinase.

• In general, the kinase reaction is followed by the ligation reaction. In this case, T4 polynucleotide kinase can be incubated in ligase reaction buffer at 37°C for 30 minutes. The products after the reaction can be directly connected without changing the buffer solution and heat inactivation. If it is necessary to maintain the dephosphorylation state of other DNA fragments during ligation, it is necessary to heat inactivate T4 polynucleotide kinase before the ligation reaction.