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Terminal Deoxynucleotidyl Transferase (TdT)

No Product Name Cat.# Conc Size Price(₩) Add
1 Terminal Deoxynucleotidyl Transferase (TdT) M020S 20 units/μl 500 units ₩ 70,000 추가
2 Terminal Deoxynucleotidyl Transferase (TdT) M020L 20 units/μl 2,500 units ₩ 280,000 추가
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Product description

Terminal deoxynucleotidyl transferase (TdT), also called terminal transferase, is a template-independent DNA polymerase that catalyzes the addition of nucleotides to the 3'-OH terminus of DNA, accompanied by the release of inorganic phosphate. TdT acts on single-stranded DNA, including 3’ overhangs of double-stranded DNA. TdT does not contain a 5’ or 3’ exonuclease domain. A gene encoding Terminal deoxynucleotidyl transferase (TdT) is cloned and expressed in E. coli, and the recombinant TdT is purified to homogeneity.

 

Characteristics

- Molecular weight: 58 KDa

- Optimal temperature: 37

- Heat inactivation: 75 for 20 min.

 

Applications

- labeling of the 3´ termini of DNA.

- Addition of homopolymer tails to the 3´ ends of DNA

- DNA sequencing

 

Quality control

- Purity: >99% on SDS-PAGE

- Endonuclease-free

- Exonuclease-free

            

Unit Definition

One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of dATP residues into DNA in 60 minutes at 37 under standard assay conditions.

 

Storage

50 mM KP04 (pH 7.3), 100 mM NaCl, 1.43 mM 2-mercaptoethanol, 0.1% Triton X-100, 50% glycerol.

 

10X Terminal Deoxynucleotidyl Transferase buffer

500 mM potassium acetate (pH 7.0), 200 mM Tris-acetate, 100 mM magnesium acetate.

 

Cautions

-TdT activity requires divalent cations.

-TdT is inhibited by metal chelators (EDTA)

 

Standard reaction conditions

A) A Typical DNA Tailing Reaction

DNA (10 pmols DNA ends*)

X μl

10X TdT Buffer

5 μl

10 mM dNTP (α-32P dATP may also be used)

0.5 μl

10X CoCl2

5 μl

Terminal Deoxynucleotidyl Transferase

0.5 μl

Distilled water

up to 50 μl

- To determine approximate amount of DNA (ng/pmol), multiply the number of base pairs by 0.66.

Example: 300 bp x 0.66 = 198 ng/pmol. For 5.0 pmols multiply by 5, resulting in 990 ng/5 pmol.

- The table below can be used as a guide (values are approximate and are given for a 30 minutes incubation at 37°C in the recommended buffer).

- The rate of addition of dNTP's and thus the length of the tail is a function of the ratio of 3´ DNA ends: dNTP concentration, and also which dNTP is used.

 

DNA Tailing Guide: 

pmols 3' ends : pmols dNTP

Tail Length

dA

dC

dG

dT

1:100

1-5

1-3

1-3

1-5

1:1,000

10-20

10-20

5-10

10-20

1:5,000

100-300

50-200

10-25

200-300

 

→ Incubate at 37°C for 30 minutes.

→ Stop the reaction by heating to 70°C for 10 minutes or by adding 10 µl of 0.2 M EDTA (pH 8.0).

 

B) A-Tailing with Terminal Deoxynucleotidyl Transferase

PCR-amplified DNA

X μl

10X TdT Buffer

7.5 μl

1 mM ddATP

1.5 μl

10X CoCl2

7.5 μl

Terminal Deoxynucleotidyl Transferase

6 μl

Distilled water

up to 75 μl

→ Incubate the reaction at 37 for 1.5 hr.



Figure 1. Enzynomics TdT is more processive than N-company.

TdT (20 and 200 ng) is incubated at 37°C for 30 min in the presence of 15 fmol of 5’ label 52 nt ssDNA (shown bottom of the figure). The asterisk in the substrate indicates the position of 32 P-label. Two different batches of TdT were compared with N-company product. Reactions were terminated by adding 4 μl of 6X stop solution (40% sucrose, 60 mM EDTA, 1.2% SDS, 0.05% bromphenol blue, and 0.05% xylene cyanol). Reaction products formed (green and red box) were separated on 12% denaturing gel in 1X TBE for 30 min at 600 V.

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