Terminal Transferase (TdT)

Description

Terminal transferase (TdT) is a template independent polymerase that catalyzes the addition of deoxynucleotides to the 3´ hydroxyl terminus of DNA molecules. Protruding, recessed or blunt ended double or single stranded DNA molecules serve as a substrate for TdT. Finnzymes´ TdT is isolated and purified from an E. coli strain carrying the cloned terminal transferase gene from calf thymus.

Applications

• Addition of homopolymeric tails to plasmid DNA and to cDNA
• Double- or single-stranded DNA 3´-termini labeling with radioactively labeled or non-radioactively labeled nucleotides
• Addition of single nucleotides to the 3´ ends of DNA for in vitro mutagenesis
• Production of synthetic homo- and heteropolymers
• RACE (Rapid Amplification of cDNA Ends)
• In situ Localization of Apoptosis
• Resolving gel compressions and artifact banding in DNA sequencing

Advantages

• Cloned and produced in E.coli
• Excellent stability and purity compared to native TdT
• Economical

Contents

TdT is supplied with optimized 10x buffer (without CoCl₂) and 25 mM CoCl₂ separately. To obtain 1 ml 1x assay buffer, mix 100 µl 10x buffer and 60 µl 25 mM CoCl₂ with 840 µl H₂O. 1x TdT Buffer contains: 20 mM Tris acetate pH 7.9 at 25°C, 50 mM Potassium acetate, supplement with 1.5 mM CoCl₂.

Storage buffer

60 mM KPO₄, 150 mM KCl, 1 mM β-mercaptoethanol, 0.5% Triton® X-100, 50 % glycerol (v/v), pH 7.2 at 25°C.

Quality Control

Unit definition
One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol dATP into acid-precipitable material in one hour at 37°C in the activity assay conditions in 1 ml volume, using (dA)₁₈ as primer.

Activity assay conditions
200 mM sodium cacodylate, 25 mM Tris-HCl (pH 7.2), 8 mM MgCl₂, 0.33 mM ZnSO₄, 0.2 mM dATP, 42 pmol oligo(dA)₁₈ and 1 µCi [³H]-dATP (0.4-1 mM) in a 50 µl total reaction volume.

Exonuclease activity
Incubation of 50 U for 4 hours at 37°C in 50 µl assay buffer with 1 µg sonicated [³H]-DNA (2 x 10⁵ cpm/µg) released <0.5 % of radioactivity.

Endonuclease activity
Incubation of 50 U with 1 µg φX174 RFI DNA (4 hours, 37°C, 50 µl) gave <10 % conversion to RFII.

Storage and stability

Stable for one year from the assay date. Recommended storage temperature -20°C.

Ordering information