Spermidine → Digoxigenin

Participants

Reaction Steps

Transcription reactions were performed using the DIG RNA Labeling Kit (Roche Molecular Biochemicals, Indianapolis, Ind.). Transcription reactions consisted of 300 ng of purified PCR product, 10×Transcription Buffer (400 mM Tris-HCl pH 8.0; 60 mM MgCl2, 100 mM dithioerythritol, 20 mM spermidine, 100 mM NaCl, 1 unit/ml RNase inhibitor), DIG Labeling mix (10 mM each of ATP, CTP, GTP, and 6.5 mM UTP, 3.5 mM UTP-DIG; in Tris-HCl pH 7.5), T7 or T3 polymerase (20 units/μl) (Promega, Madison, Wis.), and molecular grade water. Reactions were carried out at 37° C. for 2 hours, stopped with 0.2 M EDTA, pH 8.0, ethanol precipitated, and resuspended in molecular grade water to a final concentration of approximately 1 μg/μl and stored at −20° C. until use.

Provenance & Evidence