KAPA2G Fast Multiplex Kits

Figure 2. 12-plex Multiplex PCR performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I Kits. Achieving uniform representation of all amplicons in a complex multiplex assay is a challenge due to amplification bias, a result of differences in amplicon length, secondary structure and priming efficiency. Reactions (25 μL) contained 1X PCR Master Mix (KAPA and Competitor Q) or 1X PCR Buffer, 3 mM MgCl2, 0.2 mM of each dNTP and 1 U of hot-start Taq DNA Polymerase (home brew multiplex reagents, with Competitor I). Human genomic DNA was used as template (250 - 2 ng per reaction), and primers were supplied at 0.2 μM each. Cycling was performed according to manufacturers’ recommendations (30 cycles).

Figure 4. 6-plex GC-rich Multiplex PCR performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I Kits. Successful multiplex PCR with wild-type Taq is limited to easy, simple targets that can be amplified with equal efficiency. The improved processivity of the engineered KAPA2G Fast DNA Polymerase allows for uniform multiplex PCR of a broad range of difficult targets. Reactions (25 µL) contained 1X PCR Master Mix (KAPA and Competitor Q) or 1X PCR Buffer, 3 mM MgCl2, 0.2 mM of each dNTP and 1 U of hot start Taq DNA Polymerase (home-brew multiplex reagents, with Competitor I). Human genomic DNA was used as template (250 ng - 2 ng per reaction), and primers were supplied at 0.2 μM each. DMSO (5%) and KAPA Enhancer 1 (1X) were added to all reactions. Cycling was performed according to manufacturers’ recommendations (30 cycles). Amplicons range in size from 241 – 642 bp, and in GC content from 72.7 - 83.8%.