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  • Terminal Transferase

    Description

    Terminal transferase (TdT) is a template independent polymerase that catalyzes the addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules. Protruding, recessed or blunt-ended double or single-stranded DNA molecules serve as a substrate for TdT. The 58.3 kDa enzyme does not have 5' or 3' exonuclease activity. The addition of Co2+ in the reacton makes tailing more efficient.

    Highlights

    • Isolated from a recombinant source
    • Labeling of the 3' ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP)
    • Supplied with 10X Reaction Buffer and 2.5mM CoCl2

    Product Source

    An E. coli strain that carries the cloned Terminal Transferase gene from calf thymus.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    CoCl210X
    Terminal Transferase Reaction Buffer10X

    Advantages and Features

    Applications

    • Addition of homopolymer tails to the 3' ends of DNA
    • Labeling the 3' ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP)
    • TUNEL assay (in situ localization of apoptosis)
    • TdT dependent PCR

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol dATP into acid-insoluble material in a total reaction volume of 50ul in 1 hour at 37°C using d(A)18 as primer.

    Reaction Conditions

    1X Terminal Transferase Reaction Buffer
    Supplement with 0.25 mM CoCl2
    Incubate at 37°C

    1X Terminal Transferase Reaction Buffer:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    50 mM KPO4
    100 mM NaCl
    1.43 mM β-ME
    50% Glycerol
    0.1% Triton® X-100
    pH 7.3 @ 25°C

    Heat Inactivation

    75°C for 20 min

    Molecular Weight

    Theoretical: 58000 daltons

    Specific Activity

    42,000 units/mg

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    No

    Strand Displacement

    No

    Unit Assay Conditions

    1X Terminal Transferase Reaction Buffer, 0.72 μM d(A)18, 0.2 mM dATP, and 1 μCi [3H]- dATP in a 50 μl total reaction volume.

    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.
    • 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.

    References

    1. Chang, L.M. and Bollum, F.J. (1986). CRC Crit. Rev. Biochem.. 21, 27-52.
    2. Roychoudhury, R., Jay, E. and Wu, R. (1976). Nucl. Acids Res.. 3, 101-116.
    3. Tu, C.P. and Cohen, S.N. (1980). Gene. 10, 177-183.
    4. Boule, J.B., Rougeon, F. and Papanicolaou, C. (2001). J. Biol. Chem. 276, 31388-31393.

    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. Can Terminal Transferase be heat inactivated?
    2. Can Terminal Transferase label the 5' end of DNA?
    3. Is Terminal Transferase supplied with dNTPs?
    4. Does Terminal Transferase require Co2+ as a divalent cation?
    5. Can just one dNTP be added to the end of DNA with Terminal Transferase?
    6. Does Terminal Transferase have a preference for one type of 3' DNA end?
    7. What are the kM values for Terminal Transferase?
    8. Will Terminal Transferase add a non-radioactively labeled dNTP?
    1. A Typical DNA Tailing Reaction
    2. A-Tailing with Terminal Transferase