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Mesophilic DNA Polymerases >
Terminal Transferase |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Addition of homopolymer tails to the 3' ends of DNA
- Labeling of the 3' ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP)
- Supplied with 10X Reaction Buffer and 2.5mM CoCl2
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. The 58.3 KDa enzyme does not have 5' or 3' exonuclease activity. The addition of Co2+ in the reacton makes tailing more efficient.
Source:
An E. coli strain that carries the cloned terminal transferase gene from calf thymus.
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
Reagents Supplied:
Terminal Transferase Reaction Buffer
CoCl2 (10X)
Enzyme Properties
Polymerase Properties | Thermophilic Polymerase Characteristics
3´ to 5´ Exonuclease: No
5´ to 3´ Exonuclease: No
Strand Displacement: No
Heat Inactivation:
75°C for 20 minutes
Molecular Weight:
Theoretical: 58,000 daltons
Specific Activity:
42,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X Terminal Transferase Reaction Buffer
Supplemented with 0.25 mM CoCl2
Incubate at
37°C.
1X Terminal Transferase Reaction Buffer:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 1 nmol dATP into acid-insoluble material in 1 hour at 37°C using d(A)18 as primer.
Unit Assay Conditions:
1X Terminal Transferase Reaction Buffer, 0.72 μM d(A)18, 0.2 mM dATP, and 1 μCi [3H]- dATP in a 50 μl total reaction volume.
Concentration:
20,000 units/ml
Storage Conditions:
50 mM KPO4
100 mM NaCl
1.43 mM 2-Mercaptoethanol
50% Glycerol
0.1% Triton X-100
pH 7.3 @ 25°C
Storage Temperature:
-20°C
Notes
Application notes:- A Typical DNA Tailing Reaction:
1. Mix:
a. 5.0 µl 10X NEBuffer 4
b. 5.0 µl 2.5 mM CoCl2 solution provided
c. 10 pmols DNA ends, 10-100 ng depending on length
d. 1 µl of 10 mM dNTP (alpha-32P dATP may also be used)
e. 1 µl Terminal Transferase (20 units/µl)
f. H20 to a final volume of 50 µl
2. Incubate at 37°C for 30 minutes.
3. Stop the reaction by heating to 70°C for 10 minutes or by adding 10 µl of 0.2 M EDTA (pH 8.0).
FAQs
- Can Terminal Transferase be heat inactivated?
- Can Terminal Transferase label the 5' end of DNA?
- Is Terminal Transferase supplied with dNTPs?
- Does Terminal Transferase require Co2+ as a divalent cation?
- What is the rate of addition of dNTPs to the 3´ ends of DNA using Terminal Transferase?
- Can Terminal Transferase work on RNA?
- Can just one dNTP be added to the end of DNA with Terminal Transferase?
- Does Terminal Transferase have a preference for one type of 3' DNA end?
- Will Terminal Transferase add a non-radioactively labeled dNTP?
- What are the kM values for Terminal Transferase?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of Terminal Transferase with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.5% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of Terminal Transferase with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
References
- Chang, L.M. and Bollum, F.J. (1986) CRC Crit. Rev. Biochem., 21, 27-52.
- Roychoudhury, R., Jay, E. and Wu, R. (1976) Nucl. Acids Res., 3, 101-116.
- Tu, C.P. and Cohen, S.N. (1980) Gene, 10, 177-183.
- Boule, J.B., Rougeon, F. and Papanicolaou, C. (2001) J. Biol. Chem., 276, 31388-31393.
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