T4PNK-RO

T4 Polynucleotide Kinase, 3′-phosphatase free

from phage T4 am N81 pse T1 infected Escherichia coli BB

• Synonym(s): kinase, t4 polynucleotide, 3′-phosphatase-free
• UNSPSC Code: 12352200

Properties

• form: solution
• specific activity: ≥40 x 10³ units/mg protein; 40000 U/mg
• packaging: pkg of 1,000 U (10838292001); pkg of 200 U (10709557001)
• manufacturer/tradename: Roche
• storage temp.: −20°C

Description

General description

T4 polynucleotide kinase catalyzes the transfer of the terminal phosphate group of ATP to the 5′-hydroxyl terminus of DNA or RNA. It also can catalyze the exchange of 5′-terminal phosphate groups. In contrast to the wild type E. coli enzyme, this phage derivative lacks 3′-phosphatase activity. The assay time is 30 minutes and volume activity is 10000 U/ml.

Application

Use T4 Polynucleotide Kinase, 3′-phosphatase-free, to:

• Phosphorylate the 5′ ends of RNA or DNA
• Phosphorylate the 3′ ends of RNA to prevent cyclization
• Add a 5′[32P]-terminal label to 3′-CMP, forming 5′[32P]pCp. This substrate is commonly used for 3′-end labeling of RNA with T4 RNA ligase.
• Label 5′-hydroxyl ends of DNA with [?-32P]-ATP (either by direct phosphorylation or by phosphate exchange)
• Phosphorylate the 5′-hydroxyl ends of oligonucleotides obtained from automated synthesizers
• Phosphorylate the 5′-hydroxyl ends of linkers.
• Purify recombinant RNA in E. coli.[1]

Note: The 3′-phosphatase activity of the wild-type kinase is not observed under optimal incubation conditions with the mutant T4 Polynucleotide Kinase.

Biochem/physiol Actions

T4 polynucleotide kinase catalyzes the transfer of the terminal phosphate group of ATP to the 5′-hydroxyl terminus of DNA or RNA. It also can catalyze the exchange of 5′-terminal phosphate groups.[2] In contrast to the wild type E. coli enzyme, this phage derivative lacks 3′-phosphatase activity. The assay time is 30 minutes and volume activity is 10000 U/ml.

Packaging

1 kit containing 2 components

Quality

Absence of 3′-phosphatase, endonucleases, nicking activity, 3′-exonucleases, and ribonucleases, tested according to the current Quality Control procedures; function tested using the DNA 5′-End Labeling Kit.

Unit Definition

One unit is the enzyme activity which catalyzes the incorporation of 1 nmol ³²P into acid-precipitable products within 30 minutes at +37 °C.

Volume Activity: 10 x 10³ U/ml

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit Components Only

• Enzyme Solution, in buffer, pH 7.5 (+25 °C) 10 U/μl
• Phosphorylation Buffer 10x concentrated

Safety Information

• Storage Class Code: 12 - Non Combustible Liquids
• WGK: WGK 1
• Flash Point(C): does not flash