General protocol
Kit components
KAPA2G Fast Multiplex Mix (2X) is a ready-to-use cocktail containing all components for Fast Multiplex PCR, except primers and template. The Multiplex Mix contains KAPA2G Fast HotStart DNA Polymerase (1 U per 25 μl reaction), KAPA2G Buffer A (1.5X at 1X), dNTPs (0.2 mM each dNTP at 1X), MgCl2 (3.0 mM at 1X) and stabilizers.
1. Reaction setup
A typical Multiplex PCR with the KAPA2G Fast Multiplex PCR Kit consists of the following:
| Final Concentration | Volume in a 25 μl reaction | |
| PCR grade water | – | Up to 25.0 μl |
| KAPA2G Fast Multiplex Mix (2X) | 1X | 12.5 μl |
| Forward primer (10 μM) | 0.20 μM | 0.50 μl |
| Reverse primer (10 μM) | 0.20 μM | 0.50 μl |
| Template DNA | As needed | 10 – 250 ng |
2. Cycling parameters
The recommended cycling protocols for Fast Multiplex PCR with the KAPA2G Fast Multiplex PCR Kit are outlined in the table below:
| CYCLING STEP | LOW PLEX ≤5 amplicons, ≤1000 bp ≤10 amplicons, ≤500 bp |
MEDIUM PLEX ≤10 amplicons, ≤1000 bp ≤20 amplicons, ≤500 bp |
HIGH PLEX ≤10 amplicons, ≤1500 bp ≤30 amplicons, ≤1000 bp |
| Initial denaturation | 3 min at 95 °C | 3 min at 95 °C | 3 min at 95 °C |
| Denaturation | 15 sec at 95 °C | 15 sec at 95 °C | 15 sec at 95 °C |
| Annealing (see Note 1) | 30 sec at 60 °C | 30 sec at 60 °C | 30 sec at 60 °C |
| Extension (see Note 2) | 15 – 30 sec at 72 °C | 30 – 60 sec at 72 °C | 60 – 90 sec at 72 °C |
| No. of cycles (see Note 3) | 30 | 30 | 30 |
| Final extension (optional) (see Note 4) | 1 – 10 min at 72 °C | 1 – 10 min at 72 °C | 1 – 10 min at 72 °C |
NOTE:
1. Annealing should be performed for 30 sec/cycle. Start with an annealing temperature of 60 °C.
2. 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).
3. Do not perform more than 30 cycles, as this results in uneven amplification of fragments.
4. Final extension is only required if fragments are to be TA-cloned, or if reaction products will be analyzed by fluorescent capillary electrophoresis.
3. Results
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| Multiplex PCR (4- and 8-plex) performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I. 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. Cycling was performed according to manufacturers’ recommendations (30 cycles). |