KAPA2G Fast Multiplex PCR Kits

The KAPA2G Fast Multiplex PCR Kit is ideally suited for end-point, fast multiplex PCR of multiple DNA fragments, ranging in size from 50–1500 bp. This kit is ideally suited for:

• Typing of transgenic organisms
• Amplification of microsatellites
• Typing and detection of pathogens
• Amplification of multiple DNA fragments for SNP genotyping

KAPA2G Fast HotStart DNA Polymerase is the enzyme used in the KAPA2G Fast Multiplex PCR Kit. It is an antibody-mediated hot-start formulation of KAPA2G Fast DNA Polymerase, a second-generation enzyme engineered via directed evolution. It was optimized for high processivity and speed, offering faster extension rates than wild-type Taq DNA polymerase.

The KAPA2G Fast Multiplex PCR Kit offers faster and more efficient multiplex PCR than competitor enzymes based on wild-type Taq DNA polymerase. The increased speed and processivity of the engineered KAPA2G Fast HotStart DNA Polymerase result in a significant decrease in cycling times whilst maintaining the ability to amplify difficult fragments efficiently. The uniquely formulated KAPA2G Fast Multiplex Mix facilitates primer annealing and highly specific amplification of a wide range of amplicons sizes and GC content, resulting in more even amplification of all target fragments

Reaction set-up:

• Amount of starting template: Multiplex PCR assays typically require higher amounts of template than standard PCR assays. Start with at least 10 ng of genomic DNA per reaction, and at least 1 ng of less complex DNA (e.g. plasmid, lambda).
• Primer concentration: Start with 0.2 µM of each primer. Some primer sets amplify less efficiently than others and would benefit from increased primer concentration. Do not use excessive primer concentrations unnecessarily, as this may promote the formation of primer-dimer and other non-specific reaction products.
• Magnesium concentration: The KAPA2G Fast Multiplex Mix contains 3 mM MgCl₂ (at 1X), which is optimal for most assays. Additional MgCl₂ may benefit highly complex assays, as well as assays with amplicons above 1 kb.
• Amplicon length: The amplicons should not fall outside of a 1 kb range of amplicon sizes. This may result in preferential amplification of the shorter amplicons. The primer concentration of the longer amplicon may have to be increased to compensate for differences in amplification efficiency.
• Amplicon GC content: All amplicons should have a similar GC content. Multiplex PCR works best with amplicons that have a GC content of 35–55%. Amplicons with GC content ≥60% should not be combined with lower GC amplicons. For multiplex PCR of GC-rich amplicons, we recommend adding either 5% DMSO or 1M Betaine to reactions.
• Primer design and storage: All primer pairs should be validated in single reactions to confirm specificity. Primer quality is critical. Primers should be dissolved and stored in 10 mM Tris-HCl (pH 8–8.5) and never in water. Do not subject primers to multiple freeze-thaw cycles—rather keep small aliquots, or store primers at 4°C.

Cycling parameters:

• Extension time: Minimum extension time is 15 sec/cycle. Depending on the size and complexity of the multiplex assay, up to 90 sec/cycle can be used (particularly for long DNA fragments, or highly multiplexed reactions).
• Annealing time: Annealing time should be at least 30 seconds. Some assays may benefit from annealing times of up to 60 seconds, but this should be determined empirically.
• Annealing temperature: All primers in the multiplex must anneal at the same temperature, which can be determined empirically or by design. The recommended annealing temperature is 60°C, but for primers with a high Tm, an annealing temperature of up to 68°C may be used. Annealing temperatures below 60°C are not recommended as they may result in non-specific amplification.
• Cycle number: Do not perform more than 30 cycles, as this can result in uneven amplification of fragments due to reagent depletion.

Low plex (≤5 amplicons, ≤1000 bp OR ≤10 amplicons, ≤500 bp):

• Initial denaturation: 3 minutes at 95°C
• Denaturation: 15 seconds at 95°C
• Annealing: 30 seconds at 60°C
• Extension: 15–30 seconds at 72°C
• Number of cycles: 30
• Final extension: 1–10 minutes at 72°C (only required if fragments are to be TA-cloned, or if reaction products will be analyzed by fluorescent capillary electrophoresis)

Medium plex (≤10 amplicons, ≤1000 bp OR ≤20 amplicons, ≤500 bp):

• Initial denaturation: 3 minutes at 95°C
• Denaturation: 15 seconds at 95°C
• Annealing: 30 seconds at 60°C
• Extension: 30–60 seconds at 72°C
• Number of cycles: 30
• Final extension: 1–10 minutes at 72°C (only required if fragments are to be TA-cloned, or if reaction products will be analyzed by fluorescent capillary electrophoresis)

High plex (≤10 amplicons, ≤1500 bp OR ≤30 amplicons, ≤1000 bp):

• Initial denaturation: 3 minutes at 95°C
• Denaturation: 15 seconds at 95°C
• Annealing: 30 seconds at 60°C
• Extension: 60–90 seconds at 72°C
• Number of cycles: 30
• Final extension: 1–10 minutes at 72°C (only required if fragments are to be TA-cloned, or if reaction products will be analyzed by fluorescent capillary electrophoresis)

The minimum extension time is 15 sec/cycle. Depending on the size and complexity of the multiplex, up to 90 sec/cycle can be used (particularly for long DNA fragments, or highly multiplexed reactions, as shown above).

Annealing time should be at least 30 seconds. Some assays may benefit from annealing times of up to 60 seconds. The recommended annealing temperature is 60°C. For primers with a high melting temperature, an annealing temperature of up to 68°C may be used. Annealing temperatures below 60°C are not recommended, as they may result in non-specific amplification.

The KAPA2G Fast Multiplex Mix contains 3 mM MgCl₂ (1X), which is optimal for most assays. However, additional MgCl2 may benefit highly complex assays, as well as assays with amplicons above 1 kb. Any PCR-grade MgCl₂ solution may be used.

A proprietary antibody inactivates the polymerase until the first cycle of thermal denaturation. This eliminates spurious amplification products resulting from non-specific primer events during reaction setup and initiation, and improves overall reaction efficiency.

The recommended temperature for long-term storage of KAPA2G Fast Multiplex PCR Kit is -20°C. However, these kit components may be stored at 4°C for short-term usage (up to one month). If a kit component has been left on the bench for up to 72 hours, it will typically still be functional, although this storage is not recommended.

Yes, for multiplex PCR of GC-rich amplicons (60–70% GC), we recommend adding either 5% DMSO or 1M Betaine to reactions.

Up to 30 different primer pairs may be combined into a single assay using the protocols provided in the User Guide (and referenced above).