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Thermo Scientific™ Terminal Deoxynucleotidyl Transferase (TdT)


Terminal Deoxynucleotidyl Transferase (TdT) catalyzes the template-independent addition of deoxyribonucleotides to the 3'-OH terminus of DNA molecules.

Manufacturer: thermo scientific™  EP0162

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Catalog No. FEREP0162

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Description & Specifications

Specifications

Concentration 20U/µL
Inhibitors metal chelators, ammonium, chloride, iodide, phosphate ions
Source E. coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase
Units 2,500U

Terminal Deoxynucleotidyl Transferase (TdT), a template-independent DNA polymerase, catalyzes the repetitive addition of deoxyribonucleotides to the 3'-OH of oligodeoxyribonucleotides and single-stranded and double-stranded DNA. TdT requires an oligonucleotide of at least three nucleotides to serve as a primer. With RNA as template TdT shows variable performance which strongly depends upon the tertiary structure of acceptor RNA 3'-end and the nature of nucleotide. Generally, it is lower than using DNA as a template.

  • Incorporates modified nucleotides (e.g., fluorescein-, biotin-, aminoallyl-labeled nucleotides) Source: E. coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase
Quality Control:
  • The absence of endo-, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests

Source:

  • E.coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase
Definition of Activity Unit:
  • One unit of the enzyme catalyzes the incorporation of 1nmol of deoxythymidylate into a polynucleotide fraction (adsorbed on DE-81) in 60 min. at 37°C
  • Enzyme activity is assayed in the following mixture:
    • 200mM potassium cacodylate (pH 7.2), 1mM CoCl2, 0.01% (v/v) Triton X-100, 10μM oligo(dT)10, 1mM dTTP and 0.4MBq/ml [3H]-dTTP
    Storage Buffer:
    • The enzyme is supplied in:100mM potassium acetate (pH 6.8), 2mM 2-mercaptoethanol, 0.01% (v/v) Triton X-100 and 50% (v/v) glycerol

    5X Reaction Buffer:

    • 1M potassium cacodylate, 125mM Tris, 0.05% (v/v) Triton X-100, 5mM CoCl2 (pH 7.2 at 25°C)

    Inhibition and Inactivation:

    • Inhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions
    • Inactivated by heating at 70°C for 10 min. or by addition of EDTA

    Recommended for:

    Production of synthetic homo- and heteropolymers (1); Homopolymeric tailing of linear duplex DNA with any type of 3'-OH terminus (2, 3); Oligodeoxyribonucleotide and DNA labeling (2, 4-8); 5'-RACE (Rapid Amplification of cDNA Ends) (9); In situ localization of apoptosis (10)

    Note:

    Due to the presence of CoCl2 the TdT Reaction Buffer is incompatible with downstream applications. It is necessary to remove CoCl2 from the reaction mixture by spin column or phenol/chloroform extraction and subsequent ethanol precipitation.