Reaktion #1677493

ord-0c084c41e25d412587e8f744f87f5241

Reaktionsgleichung

Nc1ccn([C@H]2C[C@H](O)[C@@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)O2)c(=O)n1
dCTP
C[As](C)(=O)[O-]
cacodylate
O[C@H](CS)[C@H](O)CS
DTT
O=C(O)CN(CCN(CC(=O)O)CC(=O)O)CC(=O)O
EDTA
→
Nc1ccn([C@H]2C[C@H](O)[C@@H](COP(=O)(O)O)O2)c(=O)n1
dCMP

Edukte

Nc1ccn([C@H]2C[C@H](O)[C@@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)O2)c(=O)n1
dCTP
C[As](C)(=O)[O-]
cacodylate
O[C@H](CS)[C@H](O)CS
DTT
—
CoCl2
—
deoxynucleotide
—
10
O=C(O)CN(CCN(CC(=O)O)CC(=O)O)CC(=O)O
EDTA

Reagenzien

Keine

Lösungsmittel

O
H2O

Reaktionsbedingungen

Detaillierte Bedingungen
See reaction.notes.procedure_details.

Aufarbeitung

  1. 1
    workup.ADDITIONThe following were added together, in order

Vorschrift

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)

Quelle

DOI: 10.6084/m9.figshare.5104873.v1Patent: US05202430uspto-grants-1993_04