Non disponible en dehors du Royaume-Uni et de l'Irlande

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 fragmentsLabeling 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.

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.

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.

Packaging

1 kit containing 3 components

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)

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.

Quality

Absence of 5′ and 3′ exonucleases, endonucleases, and nicking activities tested according to the current Quality Control procedures.

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 Materials Double- or single-stranded DNA fragments Double- or single-stranded oligonucleotides