KAPA PROBE FORCE qPCR Kits

Evolved to break through.

KAPA PROBE FORCE is a highly inhibitor resistant qPCR master mix that removes the need for DNA purification, enabling streamlined sample-to-Cq workflows. The master mix contains a third generation DNA polymerase evolved to overcome blood, tissue, and plant PCR inhibitors. Crude samples can now be analyzed with comparable accuracy, reproducibility, and sensitivity as purified DNA using KAPA PROBE FORCE.

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

Are you interested in direct and crude sample extractions?

Read our new application note, “Rapid qPCR analysis from highly inhibited tissue and blood sample extractions with KAPA PROBE FORCE qPCR Kits and KAPA Express Extract.

Download our App Note


For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

Streamline sample-to-Cq workflows  in <1 hour

  • Eliminate the time and cost of sample purification by amplifying directly from crude samples
  • Analyze a wide range of sample types including  whole blood,  cells, mouse tails, FFPE, leaf, stem, seed, and soil

Generate accurate and reproducible results

  • Kits include a third-generation DNA polymerase, evolved for robust target amplification and detection
  • Enzyme maintains 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
  • 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

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Applications
  • GMO testing
  • Mouse transgenics
  • SNP genotyping
  • Food/water pathogen detection
  • Infectious disease research
  • Cancer research
Kit Specifications and Contents / Storage

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

Contents

Component
Universal (ROX pre-mixed)
FAQs
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 chemestries 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.

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.

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.

Ordering
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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for pricing