To demonstrate the validity of Transcriptic’s Autoprotocol-inplemented qPCR Protocol.
Amplification primers were generated using the Oligosynthesis Quick Instruction. Primers amplified a 265 bp amplicon encoded in the pUC19 plasmid (New England Biolabs). PCR-derived amplicons were concentrated and purified into distilled water (ISOLATE II PCR and Gel Kit; Bioline) and DNA content determined (NanoDrop Lite Spectrophotmeter; Thermo Scientific). DNA concentration was adjusted and serially diluted to generate four DNA standards(0.1; 0.01; 0.001; 0.0001 ng/µl). The RTqPCR Protocol was performed using 2 µl of DNA standard (12x replicates per sample), 0.5 µlforward primer, 0.5 µlreverse primer (both 10 µM), 7.5 µl SensiFAST SYBR No-ROX Mix (Bioline) made up to 15 µlwith distilled water in each well. Thermocycling conditions were 98 °C, 3 minutes; [95 °C, 15 seconds; 60 °C, 15 seconds; 72 °C, 20 seconds] x40. Fluorescent measurements were recorded each cycle immediately after extension.