This product contains a third-generation DNA polymerase, evolved to overcome the effect of PCR inhibitors. This DNA polymerase also enables very fast reaction protocols. The enzyme is combined with a proprietary antibody that inactivates the enzyme until the first denaturation step, eliminating spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency.
The KAPA PROBE FORCE qPCR Master Mix is compatible with all probe-based chemistries, including both hydrolysis and hybridization probes.
Primers and probes are generally used at a final concentration of 0.1 µM – 0.5 µM. It is suggested that you start with an initial final concentration of 0.2 µM for both. Use the lowest concentrations that still result in optimal PCR efficiency.
Primer and probe design
Size of amplicons
If probes are degraded, the fluorophore is separated from the quencher, leading to increased background fluorescence. Only use sterile buffers, water and laboratory plastics when diluting probes or primers and when setting up reactions.
Yes. KAPA PROBE FORCE qPCR Master Mix contains ROX at low concentration, to enable compatibility with a wide variety of real-time thermal cyclers.
The KAPA PROBE FORCE qPCR Master Mix contains magnesium chloride at a final concentration of 4.5 mM. This is sufficient for the vast majority of reactions. Extra magnesium should generally not be required, unless the reaction template is known to contain significant concentrations of Mg-chelating compounds (such as EDTA) and is used at high concentration in the reaction.
Start with the standard suggested primer/probe concentrations (0.2 µM) and reaction protocol. As long as the primers and probes have been designed for minimum interference between the assays, results should be satisfactory. Include multiple no-template control reactions to ensure that the primers and probes themselves do not cause spurious signal in the absence of template. Since the total primer concentration in a multiplex is several-fold higher than in a standard reaction, there is a much higher probability of non-specific interactions occurring. This may be combated by using the lowest primer/probe concentrations and shortest annealing/extension time that still results in optimal PCR efficiency. Reducing the PCR reaction volume also reduces the potential for non-specific interactions.
KAPA PROBE FORCE can tolerate up to 10 ng of heparin (0.0021 IU) in a 20 µl reaction without significant inhibition. Use 0.5 µl of a ten-fold dilution of heparin blood per 20 µl reaction as a starting point.
Use 1 µl of a ten10-fold dilution in a 20 µl reaction.
The antibody-mediated hot-start of KAPA 3G DNA Polymerase is fully deactivated after 20 seconds at 98°C, but optimal denaturation of complex templates may require up to 3 minutes or longer. Some types of crude samples, such as plant leaf discs or seed fragments, may require 10 min for best results.
KAPA PROBE FORCE performs best on the recommended fast cycling protocol, but will also give good results on a standard (slow) protocol.
The master mix, primer stock, water or PCR setup environment may be contaminated with DNA template or PCR product from a previous PCR amplification. It is also possible for primers and probes which have been poorly designed and/or synthesized to cause “false positive” results; degraded primers and probes may cause the same. Good laboratory practices should be followed to avoid DNA template contamination.
KAPA PROBE FORCE qPCR Master Mix should be stored at -20 °C for long-term storage (up to 12 months from receiving the kit). For short short-term storage it may be more convenient to store the kit at 4 °C for up to 3 months. Always protect the kit from light, as it contains ROX reference dye.