RTT-RO
Roche
Terminal Transferase
from Calf Thymus, recombinant, E. coli
Přihlásitk zobrazení cen stanovených pro organizaci a smluvních cen
About This Item
Doporučené produkty
recombinant
expressed in E. coli
Quality Level
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)
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
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/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
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)
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.
Pouze součásti soupravy
Č. produktu
Popis
- Terminal Transferase 400 U/μl
- TdT Reaction Buffer 5x concentrated
- CoCl<sub>2</sub> Solution 25 mM
signalword
Danger
Hazard Classifications
Acute Tox. 4 Inhalation - Acute Tox. 4 Oral - Aquatic Chronic 2 - Carc. 1B Inhalation - Repr. 1B
Storage Class
6.1D - Non-combustible, acute toxic Cat.3 / toxic hazardous materials or hazardous materials causing chronic effects
wgk_germany
WGK 3
flash_point_f
does not flash
flash_point_c
does not flash
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