Fig. 3

Optimized two-round ePCR effectively prevents formation of the chimeric molecules from two-plasmid template. a Agarose gel electrophoresis analysis of round #1 and round #2 ePCR products. Round #1 ePCR samples were generated using either the indicated amounts of an equimolar mixture of plasmid#1 and plasmid#2 (lanes 1–3) or water (no template control, “NTC”; lane 4) as template and primers Libr-A₁-for/Libr-rev. Round #2 ePCR products were obtained using either ¹/₁₀₀th of the purified round #1 ePCR samples (lanes 5–7) or water (no template control, “NTC”; lane 8) as template and primers Libr-P5-for/Libr-P7-rev. b Agarose gel electrophoresis analysis of products of test PCR set up using either ¹/₁₀₀th of the purified “100 pg” round #2 ePCR sample (lanes 1–4) or water (no template controls, “NTCs”; lanes 5–8) as template and the following primers: BC1/ROI1 (lanes 1 and 5), BC2/ROI2 (lanes 2 and 6), BC1/ROI2 (lanes 3 and 7) and BC2/ROI1 (lanes 4 and 8). c Agarose gel electrophoresis analysis of products of test PCR set up using either ¹/₁₀₀th of the purified “10 pg” round #2 ePCR sample (lanes 1–4) or water (no template controls, “NTCs”; lanes 5–6) as template and the following primers: BC1/ROI1 (lanes 1 and 5), BC2/ROI2 (lanes 2 and 6), BC1/ROI2 (lane 3) and BC2/ROI1 (lane 4). M stands for GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific) in (a, b, d)