P4390

Polynucleotide Kinase from T4-infected Escherichia coli

10 units/μL, buffered aqueous glycerol solution

• CAS Number: 37211-65-7
• Enzyme Commission number: 2.7.1.78 (BRENDA, IUBMB)
• MDL number: MFCD00166009
• UNSPSC Code: 12352204
• NACRES: NA.53

Properties

• grade: for molecular biology
• Quality Level: 200
• form: buffered aqueous glycerol solution
• mol wt: 33 kDa
• concentration: 10 units/μL
• foreign activity: Endonuclease and exonuclease, none detected
• shipped in: wet ice
• storage temp.: −20°C

Description

Application

Suitable for:

• Sequencing or nucleic acid tagging (DNA and RNA) by 5′-end labeling
• 5′ phosphorylation of oligonucleotides
• Removal of 3′-phosphate groups from phosphorylpolynucleotides

Components

T4 Polynucleotide Kinase is supplied in a solution of 50% glycerol (v/v), 20 mM Tris-HCl (pH 7.5), 25 mM KCl, 2mM DTT, 0.1 mM EDTA, and 0.1 μM ATP.

Principle

Polynucleotide kinase catalyses a "forward reaction" transfer of the γ-phosphate of ATP to the 5′ hydroxyl terminus of single- and double-stranded nucleic acids (DNA and RNA) and 3′-nucleoside monophosphates. In exchange reactions containing ADP, the enzyme will catalyze the exchange of 5′-terminal phosphate groups and ATP. The 3′-phosphatase activity enables the enzyme to remove 3′-phosphoryl groups from phosphorylpolynucleotides.
1. Forward reaction: Transfer of the labeled γ-phosphate from [γ-³²P]-ATP to the free 5′-hydroxyl group of the substrate.
5′-HO-DNA + [γ-³²P]-ATP → 5′-³²PO-DNA + ADP.
Substrates that do not have a free 5′-hydroxyl require prior dephosphorylation by alkaline phosphatase.
2. Exchange reaction: First, the terminal 5′-phosphate is transferred from the substrate to ADP present in the reaction mixture. Then, the labeled γ-phosphate from [γ-³²P]-ATP is transferred to the free hydroxyl group of the substrate.
5′-PO-DNA + ADP → 5′-HO-DNA + ATP
5′-HO-DNA + [γ-³²P]-ATP → 5′-³²PO-DNA + ADP

Unit Definition

One unit catalyzes the transfer of one nanomole of ³²P to the 5′-end of micrococcal nuclease-treated DNA in 30 min. at 37 °C. Transfer is detected as incorporation into acid-insoluble material.

Analysis Note

Activity is determined in a reaction mixture containing 40 mM Tris-HCl (pH 7.5), with 10 mM MgCl2, 5 mM dithiothreitol, 0.5 mM 5′-OH polynucleotide ends, and mM [γ-32P]-ATP.

Safety Information

• Pictograms: GHS08
• Signal Word: Danger
• Hazard Statements: H334
• Precautionary Statements: P261 - P284 - P501
• Hazard Classifications: Resp. Sens. 1
• Storage Class Code: 10 - Combustible liquids
• WGK: WGK 3
• Flash Point(F): Not applicable
• Flash Point(C): Not applicable
• Personal Protective Equipment: dust mask type N95 (US), Eyeshields, Gloves