This protocol is for reducing the disulfide bond of thiol-modified oligonucleotides to the active sulfhydryl form (Figure 1). Sulfhydryl groups can be used for attaching oligonucleotides to solid surfaces, such as gold nanoparticles. Reduction of the disulfide bonds using Dithiothreitol (DTT), also known as Cleland's reagent, also protects against primer dimer.
Figure 1. Example disulfide bond reduction of a 5'-Thiol-Modifier C6 S-S oligonucleotide. Thiol-modified oligonucleotides are shipped in disulfide form to prevent spontaneous, uncontrolled oxidation, which in turn would lead to dimer formation, thereby rendering the oligonucleotide useless.
The method is divided into two main steps: 1) sulfhydryl formation, and 2) byproduct removal.
Prepare sodium phosphate buffer, both at pH 8.3 - 8.5 and 6.0 according to standard laboratory practices. Make all buffers with Milli-Q® water.