Multiplex logic processing isothermal diagnostic assays for an evolving virus

Sanchita Bhadra; Miguel A. Saldaña; Hannah Grace Han; Grant L. Hughes; Andrew D. Ellington

Denaturing 8% polyacrylamide gels containing 7 M urea were prepared using 40% acrylamide and bis-acrylamide solution, 19:1 (Bio-Rad) in 1X TBE buffer (89 mM Tris Base, 89 mM Boric acid, 2 mM EDTA, pH 8.0) containing 0.04% ammonium persulphate and 0.1% TEMED. An equal volume of 2X denaturing dye (7 M urea, 1X TBE, 0.1% bromophenol blue) was added to the RNA samples. These were incubated at 65 °C for 3 min followed by cooling to room temperature before electrophoresis. RNA bands were demarcated using UV shadowing. Desired bands were excised from the gel and the RNA was eluted twice into TE (10:1, pH 7.5) buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0) by incubation at 70 °C and 1000 rpm for 20 min. Acrylamide traces were removed by filtering eluates through Ultrafree-MC centrifugal filter units (EMD Millipore, Billerica, MA, USA) followed by precipitation with 2X volume of 100% ethanol in the presence of both 15 µg GlycoBlue (Life Technologies) and 0.3 M sodium acetate, pH 5.2. RNA pellets were washed once in 70% ethanol. Dried pellets of purified RNA were resuspended in 0.1 mM EDTA and stored at -80 °C.