Attention:

Certain features of Sigma-Aldrich.com will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

Terminal Transferase from calf thymus

Catalog Number: T4427
Storage Temperature –20 °C

CAS RN 9027-67-2
EC 2.7.7.31
Synonyms: TdT: Terminal Deoxynucleotidyl
Transferase

Product Description

Terminal transferase is a unique DNA polymerase. In the presence of a divalent cation, it catalyzes the addition of deoxynucleotides to the 3'-hydroxyl terminus of DNA with the release of inorganic phosphate. The enzyme demonstrates a preference for a 3' overhang; however, blunt or 3' recessed ends can be used, although less effectively. Mg2+ is the preferred cation when a purine (dATP or dGTP) is to be added to DNA with a 3' overhang. When a pyrimidine (TTP or dCTP) is to be added, Co2+ should be used. Blunt ended or 3' recessed DNA can be more effectively tailed using Co2+ or Mn2+.1,2

These properties have proven to be useful in a number of applications including cloning and radioactive or non-radioactive labeling of DNA:3,5,6

  • Add homopolymers to vectors, inserts and cDNA for cloning
  • Label the 3′-end of double- and single-stranded DNA with non-radioactive and radioactive labels
  • Carry out in vitro mutagenesis by adding single nucleotides to DNA
  • Use in TUNEL assays with deoxyuridine triphosphate (dUTP) conjugated with a fluorescent chromophore or biotin to label nicked DNA in apoptotic cells

The product is supplied as a solution in 50 mM potassium phosphate, pH 7.4, with 1 mM 2-mercaptoethanol and 50% glycerol (v/v).

Activity: ≥5,000 units/ml

Unit Definition: One unit will incorporate 1 nanomole of dATP into acid precipitable material in one hour at 37 °C using oligo (dT)6 as a primer.

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.

Storage/Stability

The product ships on dry ice and storage at –20 °C is recommended.

Procedure

Suggested Reaction Buffers:
For purine tailing:
100 mM potassium cacodylate, pH 7.2
4 mM MgCl2
1 mM purine (dATP or dGTP)
20-50 units of terminal transferase
1-30 pmoles of 3' ends
25-30 µl final volume
Incubate at 37 °C for 1 hour

For pyrimidine tailing:
100 mM potassium cacodylate, pH 7.2
2 mM CoCl2
1 mM pyrimidine (TTP or dCTP)
20-50 units of terminal transferase
1-30 pmoles of 3' ends
25-3 µl final volume
Incubate at 37 °C for 1 hour

Materials


     

References

  1. Deng, G-R., and Wu, R., Methods in Enzymology, 100, 96 (1983).
  2. Roychoudhury, R. et al., Nucl. Acids Res., 3, 863 (1976).
  3. Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY: 1989) p. 5.56.
  4. Tu, C.-P.O., and Cohen, S.N., Gene, 10, 177 (1980).
  5. Kokym, R., and Horth, E., Int. J. Radiat. Biol., 68, 133 (1995).
  6. Figeys, D. et al., Anal Chem., 23, p. 4382 (1994)

 

JB,KH,EWK,MAM 01/07-1

Related Links

Categories