Thermo Scientific™ Terminal Deoxynucleotidyl Transferase (20 U/μL)

Description

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

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.

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Specifications

• Concentration: 20 U/μL
• Enzyme: Terminal Deoxynucleotidyl Transferase
• Compatible Buffer: Storage Buffer, 5X Reaction Buffer
• Quantity: 2,500 units
• Product Type: TdT

For Research Use Only. Not for use in diagnostic procedures.