KAPA2G Robust PCR Kits

Overview

KAPA2G Robust PCR Kits contain a second-generation DNA polymerase engineered to solve inconsistent amplification across a broad range of amplicon types (GC- and AT-rich).  The high performance of the DNA polymerase is ideally suited for challenging PCR applications and difficult samples, eliminating the need for optimization using multiple enzymes and protocols.

• High processivity and specific activity, translating to robust PCR performance and high sensitivity
• Improved tolerance to common PCR inhibitors

Figure 1. Half of each of the PCR products obtained with 72 of the 96 primer sets used in this study were electrophoresed in a 1% TBE-agarose gel. Amplicons were loaded in order of increasing GC content, with the lowest GC content (27%, blue) at the top left hand side and the highest GC content (84%, red) at the bottom right hand side of each composite gel image. Primers selected for this study had variable primer lengths, sequence composition, theoretical melting temperatures and other design features. Some primers contained 5'-tails for post-PCR sequencing using M13 or other standard sequencing primers. KAPA2G Robust HotStart ReadyMix reactions (25 µL) were performed as outlined in the User Guide. All reactions contained 25 ng human genomic DNA. 5% DMSO was included in all reactions (KAPA2G Robust and Taq Kits) targeting amplicons with a GC content >70%.

Figure 2. Half of each of the PCR products obtained with 72 of the 96 primer sets used in this study were electrophoresed in a 1% TBE-agarose gel. Amplicons were loaded in order of increasing GC content, with the lowest GC content (27%, blue) at the top left hand side and the highest GC content (84%, red) at the bottom right hand side of each composite gel image. Primers selected for this study had variable primer lengths, sequence composition, theoretical melting temperatures and other design features. Some primers contained 5'-tails for post-PCR sequencing using M13 or other standard sequencing primers. KAPA2G Robust HotStart ReadyMix reactions (25 µL) were performed as outlined in the User Guide. All reactions contained 25 ng human genomic DNA. 5% DMSO was included in all reactions (KAPA2G Robust and Taq Kits) targeting amplicons with a GC content >70%.

Figure 3. Amplification of a 1.5 kb fragment from 1 pg plasmid DNA in the presence of four common PCR inhibitors using the KAPA2G Robust HotStart PCR kit and wild-type hot start <i>Taq</i> polymerase. All reactions contained 0.5 units of enzyme per 25 µL reaction. KAPA2G Robust HotStart Buffer B was used throughout, with the addition of KAPAEnhancer 1 for reactions containing SDS. Cycling was performed with an Eppendorf Mastercycler epgradient S, using a standard 3-step cycling profile (35 cycles) with an annealing temperature of 64ºC and 1.5 min extension time per cycle for all enzymes.

Figure 4. Amplification of a cloned 2.7 kb insert from four commonly used <i>E. coli</i> strains (DH5a, DH10B, JM109 or BL21) using KAPA2G Robust HotStart (top) or wild-type Taq (bottom). Colonies (grown on LB-agar + Amp plates) were either resuspended directly in individual PCR reactions (left) or first resuspended in PCR grade water and then added to PCR reaction mixes (middle). For overnight cultures (prepared in LB + Amp), 1 μL was added directly to the PCR mix

Figure 5. Amplification of a 2.5 kb (left) or 1.6 kb (right) fragment from the GSH1 gene from three commonly used