末端脱氧核苷酸转移酶（TdT）

描述

Description
Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyzes the addition of deoxynucleotides to the 3’ hydroxyl terminus of single or double stranded DNA molecules.  Protruding, recessed or blunt-ended double or single-stranded DNA molecules are substrates for TdT. The 58 kDa enzyme does not have 5′ or 3′ exonuclease activity. The presence of 1 mM Co2+ stimulates the tailing of the 3’-ends of DNA fragments.

Applications

• Addition of homopolymer tails to the 3′ ends of DNA
• Labeling the 3′ ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP)
• TUNEL assay (in situ localization of apoptosis)
• TdT dependent PCR

Source
An E. coli strain that carries the cloned Terminal transferase gene from calf thymus.

Purity
> 99 % as determined by SDS-PAGE

Unit Definition
One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol dTTP into acid-insoluble material in a total reaction volume of 50 µl in 1 hour at 37°C using d(A)18 as primer.

Specific Activity
42,000 units/mg

5′ – 3′ Exonuclease

No

3′ – 5′ Exonuclease
No

Storage
-20°C

Storage Buffer
100 mM potassium acetate (pH 6.8)
2 mM 2-mercaptoethanol
0.01% (v/v) Triton X-100
50% (v/v) glycerol

10X Reaction Buffer
500 mM Potassium Acetate
200 mM Tris-acetate
100 mM Magnesium Acetate (pH 7.9 @ 25°C)
25 mM CoCl2 (pH 7.2 at 25 °C)

Inhibition and Inactivation

• Inhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions, heparin, chemically modified DNA.
• Inactivated by heating at 70 °C for 10 min or by addition of EDTA.

Note
Due to the presence of CoCl2 the TdT Reaction Buffer is incompatible with downstream applications. It is necessary to remove CoCl2 from the reaction mixture by spin column or phenol/chloroform extraction and subsequent ethanol precipitation.

其他信息

Manual & Protocols