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
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
- The absence of endo-, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests
- E.coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase
- 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
- 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
- 1M potassium cacodylate, 125mM Tris, 0.05% (v/v) Triton X-100, 5mM CoCl2 (pH 7.2 at 25°C)
- Inhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions
- Inactivated by heating at 70°C for 10 min. or by addition of EDTA
5X Reaction Buffer:
Inhibition and Inactivation:
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)
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.
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|metal chelators, ammonium, chloride, iodide, phosphate ions|
|E. coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase|
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