Terminal Deoxynulceotidyl Transferase (TdT)

OEM by QIAGEN offers bulk manufacturing of Terminal Deoxynucleotidyl Transferase (TdT) in custom formulations.

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Terminal Deoxynulceotidyl Transferase (TdT) (6000 U)

Cat. No. / ID: P7070L

6000 U (evaluation pack) Terminal Deoxynucleotidyl Transferase (TdT) (0.30 mL at 20,000 U/mL) and 10x Green Buffer (1 x 1.5 mL) and 2.5 mM Colbalt Chloride (1 x 1.5 mL)
Certain applications in which this product can be used may be covered by patents issued and applicable in the United States and abroad. Purchase of this product does not include a license to perform any patented application; therefore it is the sole responsibility of the users of the product to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used.
The Terminal Deoxynulceotidyl Transferase (TdT) is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Features

  • Efficiently extends blunt, 5’ overhanging, single stranded DNA and RNA
  • Useful for 3’ labeling
  • Incorporates dATP and dTTP with a fivefold higher efficiency than dCTP and dGTP

Product Details

Terminal Deoxynulceotidyl Transferase (TdT) efficiently extends blunt, 5′-overhanging and single-stranded DNA, and RNA. Useful for 3′ labeling. this template-independent DNA polymerase catalyzes the addition of deoxynucleotides to the 3′ hydroxyl terminus of single- or double-stranded DNA molecules. The presence of 1 mM Co2+ stimulates the tailing of the 3ʹ ends of DNA fragments. This construct is sold as an N-terminal truncation of the terminal transferase gene attached to an N-terminal fusion tag.

The enzyme is supplied in 50 mM KPO4, 100 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, <0.1% Triton X-100 and 50% glycerol; pH 7.3 at 25°C.

The enzyme is provided with 10X Green Buffer (B0120) and 2.5 mM CoCl2 (B0220). 10X Green Buffer contains the following: 200 mM Tris-acetate, 500 mM potassium acetate and 100 mM magnesium acetate; pH 7.9 at 25°C.

Performance

Polymerase properties

  • Storage temperature: –25°C to –15°C
Test Units tested Specification
Purity (SDS-PAGE) n/a >99%
Specific activity n/a 27,400 U/mg
Single-stranded exonuclease 200 U <5.0% released
Double-stranded exonuclease 200 U <1.0% released
Double-stranded endonuclease 200 U No conversion
E. coli DNA contamination 200 U <10 copies

Reference:

  1. Deng, G.R., and Wu, R. (1983) Meth. Enzymol. 100:96.

Principle

Source of recombinant enzyme protein
The protein is produced by an E. coli strain that carries the cloned terminal deoxynucleotidyl transferase gene from calf thymus.

Unit definition:
One unit is defined as the amount of polymerase required to convert 1 nmol of dTTPs into acid insoluble material in 1 hour at 37°C.

Procedure

Instructions for non-templated addition of dNTPs to 3ʹ termini of DNA
Reaction setup:

Amount Description Final concentration
5 µl 10X Green Buffer (B0120) 1X
X µl 10 pmol DNA termini (10–100 ng) 1–10 ng/µl
X µl Deoxynucleotide solution 200 µM
1 µl Terminal Deoxynulceotidyl Transferase (TdT) (20 U/ µl) 30 U/µl
X µl Type I Water n/a
50 µl Total volume  
  1. Incubate at 37°C for 30 minutes.
  2. Inactivate the Terminal Deoxynulceotidyl Transferase (TdT) and stop the reaction by heating to 70°C for 10 minutes.

Notes
Co2+ increases the nucleotide incorporation efficiency of pyrimidines, and at blunt and 3ʹ recessed ends. However, the addition of dNTPs to 3ʹ-overhanging ends is more efficient than with 3ʹ -recessed or blunt ends. Terminal Deoxynulceotidyl Transferase (TdT) requires a free 3ʹ-hydroxyl group to make a non-templated nucleotide addition.

With limited efficiency, Terminal Deoxynulceotidyl Transferase (TdT) will incorporate ribonucleotides, biotinylated and dideoxynucleotides in the presence of Co2+.

Terminal Deoxynulceotidyl Transferase (TdT) incorporates dATP and dTTP with a fivefold higher efficiency than dCTP and dGTP, as evidenced by the following Km values for nucleotides:

Base Km
dATP 100 µM
dTTP 100 µM
dCTP 500 µM
dGTP 500 µM

Quality control analysis

Unit activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 50 µl reactions containing oligo dT 20mer DNA, 1X reaction buffer, 0.25 mM CoCl2 3H-dTTP and 100 µM dTTPs. Reactions were incubated 10 minutes at 37°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

Protein concentration is determined by OD280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Applications

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

  • 3ʹ labeling
  • Extend blunt 5 ʹ overhangs

Resources