Description:
DNA Homopolymeric Tailing Kit provides qualified reagents for the addition of homopolymer tails to the 3' ends of DNA with terminal deoxynucleotidyl transferase (TdT). Under optimized assay condition, approximately average of 30~70nt oligo(dA) and oligo(dT) or 15~45nt oligo(dC) and oligo(dG) could be added to the target substrate.
TdT is a template-independent DNA polymerase that catalyzes the repetitive addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules. The enzyme was generated from an E. coli strain that carries the cloned TdT gene from calf thymus with selected mutations. Protruding, recessed or blunt-ended double or single-stranded DNA molecules serve as a substrate for TdT. The addition of dNTPs to 3′-overhanging ends is more efficient than with 3'-recessed or blunt ends. TdT incorporates dATP and dTTP with higher efficiency than dCTP and dGTP. The optimized master mixture stimulates the tailing of the 3'-ends of DNA fragments, even applicable for incorporating ribonucleotides and modified nucleotides (e.g., fluorescein-, biotin-, aminoallyl-labeled nucleotides and dideoxynucleotides).

Figures:

Figure 1: Fragment analysis through 3730 xl DNA Analyzer shows an oligo(dA) tail was added to 5’ fluorescently labeled single strand DNA oligo using DNA Homopolymeric Tailing Master Mix (dA) Kit. Arrow: Tailing reaction final product. A: Fifteen minutes at 37°C reaction product with a 50 nt average length of homopolymer dA tail. B: Thirty minutes at 37°C reaction product with a 65 nt average length of homopolymer dA tail.


Figure 2: Fragment analysis through 3730 xl DNA Analyzer shows an oligo(dC) tail was added to 5’ fluorescently labeled single strand DNA oligo using DNA Homopolymeric Tailing Master Mix (dC) Kit. Arrow: Tailing reaction final product. A: Twenty minutes at 37°C reaction product with a 38 nt average length of homopolymer dC tail. B: Forty minutes at 37°C reaction product with a 45 nt average length of homopolymer dC tail.

List of Components:
DNA Homopolymeric Tailing Kit is supplied at 2X concentration to allow approximately 50% of the final reaction volume to be used for the addition of substrate solution. Reagents sufficient for 20 or 100 tailing reactions of 10 µL each are provided.
DNA Homopolymeric Tailing Kit 20-rxn size:
Tailing Enzyme 10ul supplied with 2x:
NGMA-100: Master Mix (dA) 100µL
NGMT-100: Master Mix (dT) 100µL
NGMC-100: Master Mix (dC) 100µL
NGMG-100: Master Mix (dG) 100µL

DNA Homopolymeric Tailing Kit 100-rxn size:
Tailing Enzyme 50ul supplied with 2x:
NGMA-200: Master Mix (dA) 500µL
NGMT-200: Master Mix (dT) 500µL
NGMC-200: Master Mix (dC) 500µL
NGMG-200: Master Mix (dG) 500µL

Components:
Terminal Deoxynucleotidyl Transferase, Sodium Chloride, Potassium Acetate, Tris-acetate, Magnesium Acetate, Cobalt(II) Chloride and Stabilizers, one of Deoxyadenosine, Deoxythymine, Deoxycytidine and Deoxyguanine.
 

Recommended Storage Conditions:
DNA Homopolymeric Tailing Kit should be stored at -20°C. The Master Mix should be prevented from light.

References:
1. F. J. Bollum, Terminal deoxynucleotidyl transferase, The Enzymes, the third edition (Boyer, P.D., ed.3, Academic Press, New York, vol.10, 1974) pp. 145-171.
2. G. R. Deng, R. Wu, Terminal transferase: Use in the tailing of DNA and for in vitro mutagenesis. Meth. Enzymol. 100, 96-116 (1983).