Qiagen, P7070L, Terminal Deoxynulceotidyl Transferase (TdT) (6000 U)

6000 U of 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 Cobalt Chloride (1 x 1.5 mL)

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 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. The presence of 1 mM Co 2+ stimulates the tailing of the 3’-ends of DNA fragments (1,2).

Supplied in: 50 mM KPO 4 , 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, 50% glycerol (pH 7.3 at 25°C) Supplied with: 10X Green Buffer (B0120) : 200 mM Tris-Acetate, 500 mM Potassium Acetate, 100 mM Magnesium Acetate (pH 7.9 at 25°C) B0220: 2.5 mM CoCl 2

Performance

Test: Amount tested
Purity: n/a
Specific activity: n/a
Single-stranded exonuclease: 200 U
Double-stranded exonuclease: 200 U
Double-stranded endonuclease: 200 U
E. coli DNA contamination: 200 U

- Storage temperature: –25°C to –15°C
- Molecular weight: 82.6 kDa

Principle

The protein is produced by an E. coli strain that carries the cloned terminal transferase gene from the calf thymus with an N-terminal fusion tag.

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

Components: Final Concentration
Type I Water: N/A
10X Green Buffer (B0120): 1X
2.5 mM CoCl 2 (B0220): 250 µM
10 pmol DNA termini (10-100 ng): 1-10 ng/µL
dNTP mix: 200 µM
TdT (P7070L): 0.4 U/µL
Total Volume =: 50 µL

- Set up the following reaction mixture in a total volume of 50 µL: Components Final Concentration Volume Type I Water N/A X µL 10X Green Buffer (B0120) 1X 5 µL 2.5 mM CoCl 2 (B0220) 250 µM 5 µL 10 pmol DNA termini (10-100 ng) 1-10 ng/µL X µL dNTP mix 200 µM X µL TdT (P7070L) 0.4 U/µL 1 µL Total Volume = 50 µL
- Incubate at 37°C for 1 hour.
- Inactivate TdT and stop the reaction by heating to 70°C for 10 minutes.

Usage Instructions

Non-templated addition of dNTPs to 3′ termini of DNA

Notes: Co 2+ increases the nucleotide incorporation efficiency of pyrimidines at blunt and 3' recessed ends. However, adding dNTPs to 3'-overhanging ends is more efficient than with 3'-recessed or blunt ends. TdT requires a free 3'-hydroxyl group to make a non-templated nucleotide addition.

With limited efficiency, TdT will incorporate ribonucleotides, biotins, and dideoxynucleotides in the presence of Co 2+ .

Quality Control

Unit activity is measured using a 2-fold serial dilution method. Dilutions of the enzyme were made in 1X reaction buffer and added to 50 µL reactions containing Oligo dT 20 mer DNA, 1X reaction buffer, 0.25 mM CoCl 2 3 H-dTTP and 100 µM dTTPs. Reactions were incubated for 10 minutes at 37°C, plunged on ice, and analyzed using the method of Sambrook and Russell (3).

Protein concentration (OD 280 ) is determined by OD 280 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 protein of interest band 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 exonucleas e 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 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 16S rDNA contamination is evaluated using 5 µL replicate samples of enzyme solution 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

- Labeling of 3ʹ ends with modified nucleotides
- Adding a homopolymeric tail to 3’-OH DNA ends
- Labeling blunt ends of dsDNA breaks (TUNEL assay)

This product is available for molecular biology applications such as:

References

1. Deng, G.R. and Wu, R. (1983) Meth. Enzymol., 100:96-116. 2. Roychoudhury, R. et al. (1976) Nucl. Acids Res., 3,101-116. 3. Sambrook, J. et al. (1989) Cold Spring Harbor Laboratory Press, Molecular Cloning: A Laboratory Manual., (2nd ed.), 5.40-5.43.