Multiplex DNA Immobilization Protocol

Multiplex chips^{43 (link), 44 (link)} were dried thoroughly with argon gas and ozone-cleaned for 20 minutes in an Uvo brand ozone cleaner at 20 mW. As per the previously defined protocols, clean chips were assembled on the surface of polycarbonate holders with an acrylic clamp and a Buna-N rubber gasket. The four quadrants of the chip were separated by the fastened gasket and clamp^{44 (link)}. Annealed 5'-thiol modified duplex DNA substrates (25 μL of 30 μM) were added to each quadrant of the multiplex chip. Substrates incubated for 18-20 hours on the gold surface to allow the formation of a DNA monolayer. DNA monolayers were washed with 20 mM Tris-HCl, 75 mM NaCl, pH 7.2, and subsequently backfilled with 0.1 mM 6-mercaptohexanol (Sigma) for 15-20 minutes. Monolayers were then washed 8-10 times per quadrant with 20 mM Tris-HCl, 75 mM NaCl, pH 7.2.