Hot Start PCR Protocol | SYBR Green JumpStart Taq ReadyMix S4438 | DNA Amplification

Catalog Number: KEM0032
Storage Temperature: -20 °C
Unit Size: 6,000 U

Product 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. The presence of 1 mM Co2+ stimulates the tailing of the 3'-ends of DNA fragments. This construct is sold as an N-terminal truncation of the terminal transferase gene attached to an N-terminal fusion tag.

Source of Protein

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

Reagent

Supplied at a concentration 20,000 U/mL in
50 mM KPO4, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton™ X-100, 50% glycerol, pH 7.3

Supplied with:

2.5 mM CoCl2
Catalog Number KEM0045B

10X Green Buffer
Catalog Number KEM0043B
200 mM Tris-Acetate, 500 mM potassium acetate,
100 mM magnesium acetate, 10 mM DTT, pH 7.9

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Unit Definition

1 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.

Protocol Reaction Setup*

Component Volume (µL) Final Concentration
10x Green Buffer 5 µL 1x
10 pmol DNA termini (10-100ng) X µL 1-10 ng/µL
Deoxynucleotide solution X µL 200 µM
Terminal Transferase (20 U/µL) 1 µL 0.4 U/µL
Cobalt Chloride 5 µL
0.25 mM
Sterile Water X µL N/A
Total Volume 50 µL  


*Total reaction volume can be adjusted as needed

  1. Incubate at 37 °C for 30 minutes.
  2. Inactivate the TdT and stop the reaction by heating to 70 °C for 10 minutes.

Usage Notes

Co2+ increases the nucleotide incorporation efficiency of pyrimidines, and at blunt and 3' recessed ends. However, the addition of dNTPs to 3'-overhanging ends is more efficient than with 3'-recessed or blunt ends. TdT requires a free 3'-hydroxyl group in order to make a non-templated nucleotide addition.

With limited efficiency, Terminal Transferase will incorporate ribonucleotides, biotinylated, and dideoxynucleotides in the presence of Co2+.

Terminal Transferase incorporates dATP and dTTP with a 5-fold higher efficiency than dCTP and dGTP, as evidenced by the following Km values for nucleotides:

 

Base Km Base Km
dATP 100 µM dCTP 500 µM
dTTP 100 µM dGTP 500 µM



Materials

     

 References

  1. Deng, G.R. and Wu, R., Meth. Enxymol. 100, 96-116 (1983).

 

Related Links