RTT-RO

Terminal Transferase

from Calf Thymus, recombinant, E. coli

• EC Number: 2.7.7.31 (BRENDA, IUBMB)
• NACRES: NA.21
• UNSPSC Code: 12352204
• Recombinant: expressed in E. coli

Properties

• recombinant: expressed in E. coli
• form: solution
• usage: sufficient for 20 reactions (03333566001), sufficient for 60 reactions (03333574001)
• packaging: pkg of 24,000 U (03333574001 [400 U per reaction]), pkg of 8,000 U (03333566001 [400 U per reaction])
• manufacturer/tradename: Roche
• application(s): genomic analysis
• storage temp.: −20°C

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)

Biochem/physiol Actions

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.

Features and Benefits

Incorporation of labeled or modified nucleotides
In 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

Preparation Note

Working solution: Standard Tailing reaction with radioactive nucleotides
Preparation of CoCl₂ working solution
Add in a sterile vial 10 μl double dist. water and 15 μl of the supplied 25 mM CoCl₂ solution: Final concentration: 15 mM

Preparation of radioactive labeling mix
dATP 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)

Analysis Note

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

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.

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/μl

Sample Materials

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

Kit Components Only

• Terminal Transferase 400 U/μl
• TdT Reaction Buffer 5x concentrated
• CoCl₂ Solution 25 mM

Safety Information

• signalword: Danger
• pictograms: GHS07,GHS08,GHS09
• pcodes: P201 - P261 - P273 - P280 - P308 + P313 - P391
• wgk: WGK 3
• flash_point_f: does not flash
• flash_point_c: does not flash
• hcodes: H302 + H332,H350i,H360F,H411
• Hazard Classifications: Acute Tox. 4 - Acute Tox. 4 Oral - Aquatic Chronic 2 - Carc. 1B Inhalation - Repr. 1B Inhalation
• Storage Class: 6.1D - Non-combustible acute toxic Cat.3 / toxic hazardous materials or hazardous materials causing chronic effects