反应详情

Double-stranded cDNA was homopolymer tailed essentially by the method of R. Roychoudhury and R. Wu, Meth. Enz., 65, pp. 43-62 (1980). The following were added together, in order: 3 μl dCTP32 (>800 Ci/mmol), 20 μl 10 X cacodylate buffer (1.4 mM potassium cacodylate, 0.3M Tris-hydrochloride, pH 7.6), 4 μl 5 mM DTT, 3 μl 10 mM dCTP, 2 μl 100 mM CoCl2, 12 units terminal deoxynucleotide transferase (PL Biochemicals; at least 8 units/pmol 3' end) in 1.5 μl, H2O to 200 μl final volume. The reaction was carried out at 12° for 1.5 min then stopped by adding 10 μl 0.5M EDTA This reaction resulted in an average of 15 dCMP residues added per 3' end of dsDNA, the optimal number for annealing and transformation (Peacock et al., Bioch. Biophys. Acta, 655, pp. 243-50 (1981)

1. additionThe following were added together, in order