KAPA PROBE FORCE qPCR Kits

Overview

KAPA PROBE FORCE qPCR Kit contains a highly inhibitor-resistant qPCR master mix that removes the need for DNA purification, enabling streamlined sample-to-Cq workflows. A third generation DNA polymerase engineered through our directed evolution technology imparts resistance to blood, tissue, and plant PCR inhibitors. This allows crude samples to be analyzed with comparable accuracy, reproducibility, and sensitivity as purified DNA.

Benefits of KAPA PROBE FORCE qPCR Kit

 

  • Direct qPCR from crude blood, tissue, and plant extracts
  • Sample-to-Cq workflows in <1 hour
  • High efficiency for accurate, reproducible, and sensitive result
  • Superior tolerance to carry-over inhibitors
  • Multiplex compatibility with crude extracts

Product Highlights

Generate accurate and reproducible results

  • High reaction efficiency in the presence of PCR inhibitors for reliable data generation

Break through high levels of qPCR inhibitors

  • Achieve greater levels of sensitivity  for inhibited blood, tissue, and plant samples compared to other available kits*
  • Convert purified DNA assays to crude workflows without observable Cq delays

Multiplex crude samples efficiently

  • Accelerate genotyping analysis with single reaction allelic discrimination of crude DNA extracts
  • Maximize data collection from precious samples, increase throughput, and reduce costs through multiplexing

*Data on file.

Kits can be stored for up to 12 months at -20ºC. Master mix includes the KAPA3G HotStart DNA polymerase, dNTPs (including dUTP), MgCl2, ROX reference dye, and stabilizers.

Specifications
Spec
Description
Compatible Platforms
All real-time qPCR systems regardless of ROX passive reference dye requirement
Starting Material
Crude blood, tissue, and plant DNA extractions, purified gDNA, cDNA
Input Amount Up to 200 ng
Available Kit Sizes
1 mL, 5 mL, 10 mL, 50 mL
Components
Component
Universal (ROX pre-mixed)

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Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK4300
07959320001
100 x 20 µL reactions (Universal)
1 mL
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KK4301
07959338001
500 x 20 µL reactions (Universal)
5 mL
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KK4302
07959346001
1000 x 20 µL reactions (Universal)
10 mL
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KK4303
07959354001
5000 x 20 µL reactions (Universal)
50 mL
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  • 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

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

  • 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.

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.

  • 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.

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.