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T4 Polynucleotide Kinase

Catalog # Concentration Size List Price Quantity Your Price
M0201L 2500 units $344.00
$309.60
M0201S 500 units $86.00
$77.40
Catalog # Size List Price Your Price
M0201L 2500 units $344.00
$309.60
M0201S 500 units $86.00
$77.40
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
  • 5' phosphorylation of DNA/RNA for subsequent ligation
  • End labeling DNA or RNA for probes and DNA sequencing
  • Removal of 3' phosphoryl groups
 
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Catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3´-monophosphates. Polynucleotide Kinase also catalyzes the removal of 3´-phosphoryl groups from 3´-phosphoryl polynucleotides, deoxynucleoside 3´-monophosphates and deoxynucleoside 3´-diphosphates (1).
Product Source
A E. coli strain that carries the cloned T4 Polynucleotide Kinase gene. It is purified by a modification of the method of Richardson (1).
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  T4 Polynucleotide Kinase M0201SVIAL -20 1 x 0.05 ml 10,000 units/ml
  T4 Polynucleotide Kinase Reaction Buffer B0201SVIAL -20 1 x 1 ml 10 X
  T4 Polynucleotide Kinase M0201LVIAL -20 1 x 0.25 ml 10,000 units/ml
  T4 Polynucleotide Kinase Reaction Buffer B0201SVIAL -20 1 x 1 ml 10 X
Application Features
  • End-labeling DNA or RNA for probes and DNA sequencing (2)
  • Addition of 5´-phosphates to oligonucleotides to allow subsequent ligation
  • Removal of 3´-phosphoryl groups (3)

Properties & Usage

Unit Definition

One unit of enzyme catalyzes the phosphorylation of 20 pmol of fluorescently labeled oligo in 30 min at 37ËšC.

Reaction Conditions

1X T4 Polynucleotide Kinase Reaction Buffer
Incubate at 37°C

1X T4 Polynucleotide Kinase Reaction Buffer
70 mM Tris-HCl
10 mM MgCl2
5 mM DTT
(pH 7.6 @ 25°C)

Usage Concentration

10,000 units/ml

Storage Buffer

10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.1 µM ATP
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 minutes

Companion Products
Materials Sold Separately
Notes
  • Gaps can be phosphorylated with elevated levels of ATP. Nicks are not phosphorylated efficiently. CTP, GTP, TTP, UTP, dATP or dTTP can be substituted for ATP as a phosphate donor (1).
  • Dephosphorylation prior to end-labeling can be avoided by utilizing the exchange reaction, although this results in lower specific activity labeling.
  • Sufficient incorporation levels can be attained using the supplied buffer supplemented with 100 µM ADP in the final reaction. However, higher levels of incorporation with the exchange reaction are achieved when using the buffer described in (2).
References
  • Richardson, CC. (1981). The Enzyme. 14, 299-314.
  • Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual.. 10.59-10.67,11.31-11.33.
  • Cameron, V. and Uhlenbeck, O.C. (1977). Biochemistry. 16, 5120-5126.
  • Berkner, K.L. and Folk, W.R. (1977). J. Biol. Chem.,. 252, 3176-3184.
  • Soltis, D.A. and Uhlenbeck, 0.C. (1982). J. Biol. Chem.. 257, 11332-11339.
  • Wang, L.K. and Human, S. (2001). J. Biol. Chem.. 276, 26868-26874.
  • Wang, L.K. and Shuman, S. (2002). Nucl. Acids Res.. 30, 1073-1080.
  • Vance, J.R. and Wilson, T.E. (2001). Mol. Cell. Biol. 21, 7191-7198.
  • Interthal, H. et al. (2005). J. Biol. Chem.,. 280, 36518-36528.
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Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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