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T4 Polynucleotide Kinase (3' phosphatase minus)

Catalog # Concentration Size List Price Quantity Your Price
M0236L 10000 units/ml 1000 units $689.00
$620.10
M0236S 10000 units/ml 200 units $173.00
$155.70
Catalog # Size List Price Your Price
M0236L 1000 units $689.00
$620.10
M0236S 200 units $173.00
$155.70
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
  • 5' phosphorylation of 3' phosphorylated mononucleotide to generate a substrate (pNp) that can be added to the 3' end of DNA or RNA
  • 5' end labeling of 3' phosphorylated oligos
Featured Videos
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  • Monarch_MiniprepProtocol_VideoThumb
    DNA Blunting Tutorial
  • Monarch_MiniprepProtocol_VideoThumb
    The Mechanism of DNA Phosphorylation
T4 Polynucleotide Kinase catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of double- and single-stranded DNA and RNA, as well as nucleoside 3´-monophosphates (1-5). This modified version exhibits full kinase activity with no 3´ phosphatase activity (6,7).
Product Source
An E. coli strain that carries a plasmid encoding the modified T4 Polynucleotide Kinase gene.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  T4 Polynucleotide Kinase (3' phosphatase minus) M0236SVIAL -20 1 x 0.02 ml 10,000 units/ml
  T4 Polynucleotide Kinase Reaction Buffer B0201SVIAL -20 1 x 1 ml 10 X
  T4 Polynucleotide Kinase (3' phosphatase minus) M0236LVIAL -20 1 x 0.1 ml 10,000 units/ml
  T4 Polynucleotide Kinase Reaction Buffer B0201SVIAL -20 1 x 1 ml 10 X
Features
5´ phosphorylation of DNA/RNA for subsequent ligation
End-labeling of DNA or RNA (2)
5´ phosphorylation of 3´ phosphorylated mononucleotides to generate a substrate (pNp) that can be added to the 3´ end of DNA or RNA by ligase activity (8)
5´ end labeling of 3´ phosphorylated oligos (9)

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

Unit Assay Conditions

1X T4 Polynucleotide Kinase Reaction Buffer, 66 µM [γ-32P] ATP (5 x 106 cpm/µmol), 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA.

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 Enzymes. 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, O.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, JR. and Wilson, T.E. (2001). Mol. Cell. Riot.. 21, 7191-7198.
  • Interthal, H. et al. (2005). J. Biol. Chem.. 280, 36518-36528.
Quality Control Assay
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
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]
Safety Data Sheets
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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