Terminal Deoxynucleotidyl Transferase (Tdt)

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Reagents Supplied

5X Tdt C-Tailing Reaction Buffer

Applications

• Prefered substrates are single-stranded DNA, double-stranded DNA with 3’-hydroxyl termini and oligodeoxynucleotide primers (1)
• Used for specific labeling of 3'-termini with ribonucleotides (2)
• Labels 3'-ends of DNA fragments with an [α-³²P] 3'-deoxynucleoside (3)
• Adds homopolymer tails of deoxyribonucleotides to vectors or cDNAs (4,5)

Unit Definition

One unit is the amount of enzyme required to transfer 1 nmol of dAMP from dATP to the 3' OH terminus of the oligonucleotide initiator d(A)15 37ºC for 1 hour

Heat Inactivation

15 minutes at 75ºC

Concentration

10-15 units/µl

Storage Buffer

50 mM potassium phosphate (pH 7.4) 
1 mM β-mercaptoethanol
50% (w/v) glycerol

Assay Conditions

40 mM K Cacodylate (pH 7.2)
8 mM MgCl₂
0.33 mM ZnSO₄
10 µM oligonucleotide d(A)50
10 µl bovine serum albumin buffer 
1 mM (α-³²P) dATP
Reaction volume of 60 µl

Note: All reactions should be run in polypropylene tubes

Storage Conditions

Store at -20°C
Product shipped on Dry Ice

References

(1) Chang, L.M. and Bollum, F.J. (1971) J. Biol. Chem. 246, 909-916
(2) Roychoudury, R. and Kossel, H. (1971) Eur. J. Biochem. 22. 310-320
(3) Tu, C.P.D. and Cohen, S.N. (1980) Gene 10, 177-183
(4) Roychoudhury, R., Jay, E. and Wu, R. (1976) Nucleic Acids Res. 3, 863-877
(5) Deng, G. and Wu, R. (1983) Methods Enzymol. 100, 96-116