KAPA PROBE FAST qPCR Kits

Figure 1. A total of 168 human genomic DNA samples were successfully genotyped along with 24 no-template controls using an ATP1B3 SNP genotyping assay on the ABI 7900HT real-time PCR system. Reaction volumes also included human genomic DNA (10 ng), 200 nM of each primer, and 200 nM of each hydrolysis probe (Allele X – FAM/ BHQ-1, Allele Y – VIC/ BHQ-1) using the following standard cycling protocol: 95ºC for 10 minutes followed by 40 cycles of 95ºC, 15 seconds; 60ºC, 60 seconds.

Figure 2. Results for all 5 amplicons across a 5 point dilution series were obtained when assayed in penta-plex using a fast cycling protocol. Reaction volumes also included human genomic DNA (0.39 ng – 100 ng), 200 nM of each primer, and 200 nM of each hydrolysis probe (ACTB - FAM/BHQ-1, ERBB2 - CAL Fluor Gold 540/ BHQ-1, ERBB3 - CAL Fluor Orange 560/ BHQ-2, EGFR - CAL Fluor Red 610/ BHQ-2, ACTG1 - Quasar 705/ BHQ-2) using the following fast cycling protocol: 95ºC for 3 minutes followed by 40 cycles of 95ºC, 15 seconds; 60ºC, 15 seconds.

Figure 3. Amplification plots were generated using either a standard cycling protocol (95ºC for 10 minutes followed by 40 cycles of 95ºC, 15 seconds; 60ºC, 60 seconds) or a fast cycling protocol: 95ºC for 3 minutes followed by 40 cycles of 95ºC, 3 seconds; 60ºC, 20 seconds. Reaction volumes also included human genomic DNA (10-fold dilutions over a 0.1 ng - 100 ng per reaction range), 200 nM of each primer, and 200 nM of hApoB100 (FAM/ BHQ-1) hydrolysis probe.