Terminal Transferase

Description

• General description: Terminal Transferase catalyzes the template independent addition of deoxy- and dideoxynucleoside triphosphates to the 3′-OH ends of double and single-stranded DNA fragments, and oligonucleotides. Terminal Transferase incorporates digoxigenin-, biotin-, and fluorochrome-labeled deoxy- and dideoxynucleoside triphosphates as well as radioactively labeled deoxy- and dideoxynucleoside triphosphates. The supplied 5x-concentrated reaction buffer allows the optimal tailing of all types of double-stranded DNA ends: blunt ended, with 3′ overhang, or with 5′ overhang. The highest incorporation rates are obtained with 3′ overhangs.
• Application: Use terminal transferase to add nucleotides to the 3′-OH ends of double- or single-stranded DNA fragments, for example:Tailing with dNTPs:Addition of homopolymeric tails to DNA fragments[1][2][3]Labeling of double- and single-stranded DNA and oligonucleotides with either radioactive or chemically modified nucleotides (e.g., DIG-dUTP)3′-end Labeling with ddNTPs:Labeling of double- and single-stranded DNA and oligonucleotides with either radioactive or chemically modified dideoxynucleotides (e.g., DIG-ddUTP)
• Features and Benefits: Incorporation of labeled or modified nucleotidesIn addition to standard nucleotides, terminal transferase wlll add radioactive or modified (e.g., digoxigenin-, biotin-, or fluorochrome-labeled) dNTPs or ddNTPs to DNA.
• Packaging: 1 kit containing 3 components
• Quality: Absence of 5′ and 3′ exonucleases, endonucleases, and nicking activities tested according to the current Quality Control procedures.
• Principle: Oligonucleotides are enzymatically labeled at their 3′ end using terminal transferase by incorporation of a single digoxigenin-labeled dideoxyuridine-triphosphate. Another way to label oligonucleotides is the addition of a longer nucleotide tail. For the generation of tailed oligonucleotide probes, deoxynucleotides triphosphates are used in a template independent reaction.
• Unit Definition: One unit is the enzyme activity that incorporates 1 nMol dTMP into acid-insoluble products within 30 minutes at +37 °C under assay conditions using d(pT)6 as primer. Unit assay conditions: 200 mM Potassium cacodylate, 1 mM CoCl2, 1 mM dTTP, 0.1 OD d(pT)6, 6.25 pmol 3H dTTP in a 120 μl reaction volume.Unit Assay: Unit assay conditions: 200 mM Potassium cacodylate, 1 mM CoCl2, 1 mM dTTP, 0.1 OD d(pT)6, 6.25 pmol 3H dTTP in a 120 μl reaction volume.Volume Activity: 400 U/μlSample MaterialsDouble- or single-stranded DNA fragmentsDouble- or single-stranded oligonucleotides
• Preparation Note: Working solution: Standard Tailing reaction with radioactive nucleotidesPreparation of CoCl2 working solutionAdd in a sterile vial 10 μl double dist. water and 15 μl of the supplied 25 mM CoCl2 solution: Final concentration: 15 mMPreparation of radioactive labeling mixdATP and dTTP labeling mix: mix 1 Vol. of a 2.5 mM dATP or dTTP solution with 15 volumes of double-distilled water and 4 volumes of α-32P-dATP or α-32P-dTTP (800 Ci/mmol, approx. 30 TBq/mmol).dGTP and dCTP labeling mix: mix 1 volume of a 2 mM dGTP or dCTP solution with 15 volumes of double-distilled water and 4 volumes of α-32P-dGTP or α-32P-dCTP (800 Ci/mmol, approx. 30 TBq/mmol)
• Other Notes: For general laboratory use. Double-stranded DNA may have either blunt-, 3′-protruding, or 5′-protruding ends. However, 3′-protruding ends lead to the highest incorporation rates.TdT requires an oligonucleotide of at least three bases as a primer, and single-stranded DNA is tailed more efficiently than double-stranded.