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
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:
Labeling of double- and single-stranded DNA and oligonucleotides with either radioactive or chemically modified dideoxynucleotides (e.g., DIG-ddUTP)
- Tailing with dNTPs:Addition of homopolymeric tails to DNA fragments
- Labeling of double- and single-stranded DNA and oligonucleotides with either radioactive or chemically modified nucleotides (e.g., DIG-dUTP)
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.
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)
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
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
Product No.
Description
- Terminal Transferase 400 U/μl
- TdT Reaction Buffer 5x concentrated
- CoCl<sub>2</sub> Solution 25 mM
signalword
Danger
wgk
WGK 3
flash_point_f
does not flash
flash_point_c
does not flash
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
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