Abil we09

An emulsifieroil mixture was prepared using 7% (w/w) Abil WE09 (Evonik Industries, Essen, Germany), 20% (w/w) light mineral oil (M8410; Sigma-Aldrich, St. Louis, MO), and 73% (w/w) Tegosoft DEC (Evonik Industries). Abil WE09 serves as the emulsifier, and Diehl et al. has previously demonstrated that water-in-oil emulsions generated with this mixture are stable under PCR conditions^{24 (link)}. The oil mixture was vortexed for 10 s at 3000 rpm and incubated at room temperature for 30 min before use. A 300 μL aliquot of emulsifier-oil and 50 μL of PCR solution were combined in a microcentrifuge tube and vortexed at 3000 rpm for 30 s at 10 s intervals. The resulting water-in-oil emulsion of non-uniform volume distribution was aliquoted into two or more PCR tubes (50 μL each) and heated in a C1000 Thermal Cycler (Bio-Rad) for 3 min at 95°C followed by 50 cycles of 30 s each at 95°C, 54°C, and 72°C. A schematic of the procedure is shown in Figure 1.

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) except as indicated. The DNA was synthesized by Integrated DNA Technologies (Coralville, IA). HIV-1 RNA (Group M, Subtype B) was ordered from SeraCare Life Sciences Inc. (Catalog # 0400–0078, Milford, MA), and the concentration was measured using an Abbott RealTime HIV-1 assay (Abbott Laboratories, Chicago, IL). A NASBA Kit was purchased from Life Sciences Advance Technologies Inc. (St. Petersburg, FL). Abil WE 09 and Tegosoft DEC were samples from Evonik Industries (Essen, Germany). Bovine serum albumin (BSA) was purchased from Thermo Fisher Scientific (Waltham, MA). Nanopure water (18.2 MΩ; MillporeCo., Bedford, MA, USA) was used in all experiments and in buffer preparation. qNASBA reactions were run and analyzed using a Bio-Rad CFX96 Real-Time PCR instrument (Bio-Rad Laboratories, Hercules, CA). dNASBA was performed using an Eppendorf thermal cycler with an in situ adapter. Images were acquired using a Typhoon FLA9000 imaging system (GE Healthcare, Pittsburgh, PA).