Terminal Deoxyribonucleotidyl Transferase 101
- 0.5 M Na2HPO 4.
- Competent cells of E. coli DH5t~ (Gibco-BRL, Eggenstein, Germany).
- 10 mM Tris-HC1, pH 7.2, 1 mM EDTA, 100 mM NaCI.
3.2. Methods
3.2.1. Preparation of the Tailing Buffer- Equilibrate 5 g Chelex 100 with 3M potassium acetate, pH 7. After 5
min at room temperature, remove excess liquid by passing the slurry
through a glass sintered funnel by applying water aspirator vacuum.
Wash the Chelex in the funnel with 10 mL distilled water by applying
water aspirator vacuum. - Prepare a 1.2M solution of cacodylic acid. Add KOH pellets, until a pH
of approx 7 is obtained. Adjust to pH 7.2 by the dropwise addition of
1M KOH. Dilute with distilled water to obtain a 1M stock solution. - Add the equilibrated Chelex to the potassium cacodylate solution, and
stir for about 5 min. Remove the ionic exchanger by filtration. - Prepare a 5X C-Tailing Buffer by pipeting in the following order:
a. 10 mL 1M potassium cacodylate, pH 7.2
b. 8.8 mL distilled water
c. 0.2 mL 0.1M DTT
d. 1 mL 0.1M CoC12
Store in 500-JxL aliquots at -20°C. - Prepare a 5X G-Tailing Buffer by pipeting in the following order:
a. 10 mL 1M potassium cacodylate, pH 7.2
b. 8.8 mL distilled water
c. 0.2 mL 0.1M DTT
d. 1 mL 0.1M MnC12
Store in 500-1aL aliquots at -20°C.
3.2.2. Preparation of Phosphorothioate-Containing
Homopolymers - Mix:
p(dC)10 (10 A260 U/mL) 1.5 ~L (10 I.tM 3'-OH)
5X C-tailing buffer 5 ~L,
[t~-35S]dCTP (1300 Ci/mmol, 13 gCi/~L) 2 lxL
5 mM dCTPaS 2.5 lxL
Distilled water 13 ~tL
Terminal transferase (17 U/laL) 1 laL - Incubate for 60 min at 37°C.
- Spot a 2-1aL aliquot onto a DE81-filter disk, dry under a fan, and count
the total radioactivity in a toluene-based scintiUant.