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:
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
Low plex (≤5 amplicons, ≤1000 bp OR ≤10 amplicons, ≤500 bp):
Medium plex (≤10 amplicons, ≤1000 bp OR ≤20 amplicons, ≤500 bp):
High plex (≤10 amplicons, ≤1500 bp OR ≤30 amplicons, ≤1000 bp):
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).