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  • T4 Polynucleotide Kinase

    Description

    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).

    Highlights

    • Isolated from a recombinant source
    • 5' end labeling of DNA and RNA
    • RNase and DNase free
    • Supplied with 10X Reaction Buffer

    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:

    Store at (°C)Concentration
    T4 Polynucleotide Kinase Reaction Buffer-2010X

    Advantages and Features

    Applications

    • 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 and Usage

    Unit Definition

    One Richardson unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of acid-insoluble [32P] in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X T4 Polynucleotide Kinase Reaction Buffer with 66 µM [γ-32P] ATP (5 x 106 cpm/µmol) and 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA (1).

    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 Temperature

    -20°C

    Storage Conditions

    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 min

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • Phosphatase Activity (Oligo):
      The product is tested in a reaction containing a 5'-phosphorylated oligo. After incubation there is no detectable dephosphorylation as determined by SDS-PAGE
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    References

    1. Richardson, C.C. (1981). P.D. Boyer(Ed.), The Enzymes. 14, 229-314. San Diego: Academic press.
    2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd ed.). 10.59-10.67, 11.31-11.33.
    3. Cameron, V. and Uhlenbeck, O.C. (1977). Biochemistry. 16, 5120-5126.
    4. Berkner, K.L. and Folk, W.R. (1977). J. Biol. Chem.. 252, 3176-3184.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What factors can cause incomplete phosphorylation when using T4 Polynucleotide Kinase?
    2. Can I use T4 Polynucleotide Kinase and T4 DNA Ligase in the same reaction buffer?
    3. How can the rate of phosphorylation be improved when using T4 Polynucleotide Kinase?
    4. Do I need to dephosphorylate prior to labeling?
    5. How much substrate can be phosphorylated in a standard reaction?
    6. How many units of T4 Polynucleotide Kinase should be used for a typical reaction?
    7. How do I inactivate the enzyme?
    8. How to calculate the molarity of ends?
    9. What labels can be used?
    1. Radioactive Labeling with T4 PNK or T4 PNK 3' phosphatase minus
    2. Non-radioactive phosphorylation with T4 PNK or T4 PNK 3' phosphatase minus

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