Maximizing PCR and RT-PCR Success

PCR Brochure 10/2016 17 995 bp 99 bp Supplier TI 1.25 U 2.5 U 3.75 U 5 U Standard QIAGEN Figure 18. Efficient 19-plex PCR using the QIAGEN Multiplex PCR Plus Kit. Multiplex PCR of 19 targets (99–955 bp) was performed using standard conditions for the QIAGEN Multiplex PCR Plus Kit, without further optimization (QIAGEN) or using a variety of concentrations of a hot-start DNA polymerase from Supplier AII. Analysis was performed using the QIAxcel Advanced System. The QIAGEN Multiplex PCR Plus Kit resulted in specific amplification of all targets without the need for optimization. Despite lengthy optimization using different enzyme concentrations, multiplex PCR using the kit from Supplier AII resulted in missing fragments, even when using higher concentrations. FAM PET FAM PET Type-it Microsatellite PCR Kit Supplier A FAM PET FAM PET Type-it Microsatellite PCR Kit Supplier A Figure 19. Optimization-free and reliable 13-plex STR analysis using the Type-it Microsatellite PCR Kit. Only two out of four channels (FAM™ and PET® ) of a 3730xl DNA Analyzer (Thermo Fisher Scientific Inc.), representing 6 of 13 analyzed STR loci are shown. Using the Type-it Microsatellite PCR Kit under standard conditions, all 6 STR loci are reliably amplified. Despite the increased number of cycles and preoptimized PCR conditions for the hot-start method of Supplier A – compared with 25 cycles and standard conditions for the Type-it Microsatellite Kit – several specific peaks are missing and nonspecific signals are observed (arrows). FAM PET FAM PET Type-it Microsatellite PCR Kit Supplier A

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