KAPA PROBE FORCE qPCR Kits FAQs

What are the recommended applications for KAPA PROBE FORCE?

  • Analysis of challenging sample types: crude DNA extractions from blood, tissue, plant material and environmental samples, cDNA templates/direct carry-over from cDNA-synthesis reactions)
  • Genotyping (GMO testing, mouse transgenics, SNP analysis/allelic discrimination)
  • Gene expression analysis (pathogen detection, infectious disease research, cancer research)
  • Multiplexing applications

What is the enzyme in KAPA PROBE FORCE?
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.

Which probe chemistries are compatible with KAPA PROBE FORCE?
The KAPA PROBE FORCE qPCR Master Mix is compatible with all probe-based chemistries, including both hydrolysis and hybridization probes.

What primer/probe concentration should I use in my qPCR reaction?
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.

Any suggestions for optimization of problematic assays?

Primer and probe design

  • Standard assays are typically designed with F & R primer Tm values at ~60°C and probe Tm at ~67°C – 70°C, with a standard PCR annealing/extension temperature of 60°C. Depending on the sequence(s) of interest, this “standard” design and PCR approach might not always give the best performance; also, it might not even be possible to easily design an assay that conforms to these standard parameters. Difficult assays are often those where the target sequence is located in high-GC areas. For most assays, the standard initial denaturation at 98°C for 3 min will effectively denature even high-GC areas of the DNA template, but in some cases it may also be required to raise the annealing/extension temperature above the normal 60°C, since the amplicon itself may have too much secondary structure at 60°C for efficient amplification and/or probe-binding. In a few extreme cases, depending on the characteristics of the PCR product, better results may be obtained if the in-cycle denaturation is also performed at 98°C (10 sec).

Size of amplicons

  • Typically, amplicons for probe-based qPCR are below 200 bp in length. For much longer PCR products, the annealing/extension time may have to be optimized in order to obtain optimal PCR efficiency.

Probe concentration

  • If low signal is experienced in a SNP assay, increasing the probe concentrations to 0.4 or 0.8 µM may help. As long as specificity is maintained, annealing/extension temperature may also be lowered a few degrees from the standard 60°C for some assays.

Instrument variation

  • It is worth keeping in mind during assay optimization that supposedly identical thermal cyclers often display significantly different performance characteristics and this may require, in some cases, the optimization of PCR protocols specific to individual cyclers.

What can cause high background levels fluorescence when working with probes?
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.

Does KAPA PROBE FORCE qPCR Master Mix contain a reference dye?
Yes. KAPA PROBE FORCE qPCR Master Mix contains ROX at low concentration, to enable compatibility with a wide variety of real-time thermal cyclers.

When do I need to add additional magnesium chloride to my qPCR reaction?
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.

What are the recommended reaction conditions for multiplex assays?
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.

Where do I begin with optimization of crude samples or crude extracts as PCR template?

  • The general rule when aiming to use crude samples or crude extracts as template, is to first optimize the size of crude sample or volume of crude extract in the reaction. Spike in a range of crude sample sizes or crude extract volumes/dilutions into an unrelated test qPCR (e.g. a crude plant extract into a qPCR that uses mouse DNA as template), to ascertain at what point unacceptable PCR inhibition starts to take place. Ideally, the optimal crude sample size or crude extract volume should cause no significant inhibition in the test qPCR, whilst still providing enough template DNA for reliable amplification in an assay that targets the DNA in the crude sample itself.
  •  Some crude samples, such as mouse-tail extracts, cause very low or no PCR inhibition and are very easy to incorporate into a crude-sample workflow. Others, such as crude plant extracts, typically cause more inhibition and need to be optimized more carefully.

How much heparin blood or heparin per reaction can be tolerated by KAPA PROBE FORCE?
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.

How much EDTA blood per reaction is recommended for use with KAPA PROBE FORCE?
Use 1 µl of a ten10-fold dilution in a 20 µl reaction.

Why is the recommended initial denaturation time so long (3 – 10 min)?
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.

Can I use a standard cycling protocol rather than a fast cycling protocol?
KAPA PROBE FORCE performs best on the recommended fast cycling protocol, but will also give good results on a standard (slow) protocol.

What can cause no-template controls (NTCs) to give produce a positive result?
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.

What are the storage recommendations for the KAPA PROBE FORCE qPCR Master Mix?
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.

Materials

     
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