KAPA PROBE FAST qPCR Kits

Optimized for Versatility and Speed.

KAPA PROBE FAST qPCR Kits provide fast and reproducible results for all probe-based qPCR applications. Kits contain a ready-to-use master mix for highly sensitive and accurate real-time PCR using sequence-specific fluorogenic probe chemistries, including hydrolysis probes (e.g. TaqMan®), FRET probes, and displacement probes (e.g. molecular beacons).

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

Product Highlights

A total of 168 human genomic DNA samples were successfully genotyped along with 24 no-template controls using an ATP1B3 SNP genotyping assay on the ABI 7900HT real-time PCR system. Reaction volumes also included human genomic DNA (10 ng), 200 nM of each primer, and 200 nM of each hydrolysis probe (Allele X – FAM/ BHQ®-1, Allele Y – VIC/ BHQ-1) using the following standard cycling protocol: 95ºC for 10 minutes followed by 40 cycles of 95ºC, 15 seconds; 60ºC, 60 seconds. Data on file.

Accurately discriminate between alleles

  • Exceptional differentiation of heterozygous and homozygous alleles
  • Discrete clusters in SNP genotyping assays

Achieve excellent reproducibility and efficiency

  • Fast, high performance five-colored multiplex PCR
  • Similar abundance levels achieved with low copy number
  • Broad dynamic range of up to 10 orders of magnitude
Amplification plots were generated using either a standard cycling protocol (95ºC for 10 minutes followed by 40 cycles of 95ºC, 15 seconds; 60ºC, 60 seconds) or a fast cycling protocol: 95ºC for 3 minutes followed by 40 cycles of 95ºC, 3 seconds; 60ºC, 20 seconds. Reaction volumes also included human genomic DNA (10-fold dilutions over a 0.1 ng - 100 ng per reaction range), 200 nM of each primer, and 200 nM of hApoB100 (FAM/ BHQ®-1) hydrolysis probe. Data on file.

Increase flexibility

  • Compatible with both standard and fast cycling protocols
  • Retain accurate quantification
Applications
  • Genotyping
  • Gene expression analysis
  • Multiplex qPCR
Kit Specifications and Contents / Storage

Kits can be stored for up to 12 months at -20˚C.

Kits include KAPA PROBE FAST qPCR Master Mix (2X), which contains KAPA Taq HotStart DNA Polymerase, reaction buffer, dNTPs, and MgCl2 at a final concentration of 5 mM. Where noted, Master Mixes contain instrument-specific reference dyes, while the Universal kit includes ROX High and ROX Low (both at 50X) separately.

Components

PROBE_Component Chart

Specifications

Spec
Description
Starting Material
DNA, plasmid DNA, cDNA
FAQs
What is the enzyme in KAPA PROBE FAST qPCR Master Mix (2X) Kits?

This product contains KAPA Taq HotStart DNA Polymerase. In the HotStart formulation, 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.

With which probe chemistries can KAPA PROBE FAST qPCR Master Mix Kits be used?

KAPA PROBE FAST qPCR Kit is compatible with all probe-based chemistries including both hydrolysis and hybridization probes. The kit has been optimized for optimal performance in multiplex assays, making this kit particularly well suited to gene expression analysis. This kit can also be used for allelic discrimination assays such as SNP genotyping assays.

At what concentration should I use my probes in my qPCR reaction?

Probes are generally used at a final concentration of 100 nM–400 nM. We suggest starting with 200 nM final concentration.

In which buffer should qPCR probes be resuspended?

Fluorophores are sensitive to hydrolysis, which is accelerated at low pH. We recommend resuspending probes in pH 8.0 TE Buffer (10 mM Tris-Cl, 1 mM EDTA).

How should qPCR probes be stored?

Probes should be subjected to a minimum number of freeze-thaw cycles. For this reason probes are best prepared first for long term storage at -20ºC or -80ºC as 100 μM concentrated stocks. Dilute a portion of the stock to an appropriate working concentration e.g., 10 μM, aliquot into microvials and store at -20ºC or -80ºC. To ensure optimum activity, fluorescent probes should always be protected from light to avoid photobleaching. Probes and primers stored at -20ºC or -80ºC are stable for over one year. However, please refer to your probe manufacturer’s instructions for specific information to your probe formulation.

What can cause high background levels when working with probes?

Probes are very sensitive to degradation, which separates the fluorophore from the quencher. When aliquoting fluorescently labeled probes, sterile tubes and tips must be used to avoid contamination with DNases.

At what concentration should I use my primers in my qPCR reaction?

Primers are generally used at a final concentration of 100 nM – 400 nM. We suggest starting with 200 nM final concentration.

When would I add ROX passive reference dye to my qPCR reaction?

For certain real-time cyclers, the presence of ROX reference dye in real-time PCR compensates for non-PCR-related variations in fluores­cence detection. Fluorescence from ROX reference dye does not change during the course of real-time PCR, but provides a stable baseline to which PCR-related fluorescent signals are normalized. Thus, ROX dye compensates for differences in fluorescence detection between wells due to slight variations in reaction volume or to differences in well position. The use of ROX dye is necessary for all instruments from Applied Biosystems and is optional for the Mx3000P®, Mx3005P™, and Mx4000®. Instruments from Bio-Rad/MJ Research, Cepheid, Corbett Research, Eppendorf, and Roche do not require ROX dye.

Do I need to add additional magnesium chloride to my qPCR reaction?

The concentration of MgCl2 affects the binding dynamics of primers and probes to template DNA. The higher the final MgCl2 concentration of the PCR reaction, the greater the binding affinity of the primers and probe for target DNA. The KAPA PROBE FAST qPCR Master Mix (2X) provides MgCl2 at a final concentration of 5.0 mM. It is highly unlikely that additional MgCl2 will improve reaction efficiency or specificity.

Why would I require initial activation times of greater than 10 sec at 95ºC for an antibody-mediated hot-start DNA polymerase?

Although the antibody-mediated hot-start version of KAPA Taq DNA Polymerase is activated after 10 seconds at 95ºC, optimal denaturation of template may require up to 3 minutes. Human genomic DNA, for example, would require a longer initial denaturation time than plasmid DNA.

Can I use a standard cycling protocol rather than a fast cycling protocol?

Yes, KAPA PROBE FAST has been formulated to support both standard (slow) and fast cycling protocols.

What can cause no template controls (NTC) to give a positive result?

The master mix, primer stock and/or water may be contaminated with DNA template or PCR product from a previous PCR amplificaiton. Good laboratory practices should be used to avoid DNA template contamination. It is also possible for primers and probes which have be poorly designed, synthesized or degraded to result in “false positive” results.

What are the storage recommendations for KAPA PROBE FAST qPCR kits?

KAPA PROBE FAST qPCR Kits should be stored at -20ºC for long term storage up to 1 year from receiving the kit. This kit retains stability and performance for up to 30 freeze-thaw cycles. For short term storage it may be more convenient to store the kit at 4ºC for up to 3 months. Always protect the kit from light if it contains either ROX or Fluorescein reference dyes.

Can KAPA PROBE FAST qPCR Kits be used with the Roche LightCycler® II capillary qPCR instrument?

Yes. However, the Roche LightCycler® II capillary instrument requires the addition of BSA to the qPCR reaction at a final concentration of 250 ng/µL in order to prevent the DNA polymerase and template from binding to the glass capillaries.

Ordering

Kits include KAPA PROBE FAST qPCR Master Mix (2X), which contains KAPA Taq HotStart DNA Polymerase, reaction buffer, dNTPs, and MgCl2 at a final concentration of 5 mM. Where noted, Master Mixes contain instrument-specific reference dyes, while the Universal kit includes ROX High and ROX Low (both at 50X) separately.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK4701
07959800001
100 x 20 µL reactions (Universal)
1 mL
Login
for pricing
KK4702
07959818001
500 x 20 µL reactions (Universal)
5 mL
Login
for pricing
KK4703
07959826001
1000 x 20 µL reactions (Universal)
10 mL
Login
for pricing
KK4705
07959842001
100 x 20 µL reactions, ROX (ABI Prism®)
1 mL
Login
for pricing
KK4706
07959869001
500 x 20 µL reactions, ROX (ABI Prism)
5 mL
Login
for pricing
KK4707
07959877001
1000 x 20 µL reactions, ROX (ABI Prism)
10 mL
Login
for pricing
KK4709
07959893001
100 x 20 µL reactions, with fluorescein (Bio-Rad® iCycler™)
1 mL
Login
for pricing
KK4710
07959907001
500 x 20 µL reactions, with fluorescein (Bio-Rad iCycler)
5 mL
Login
for pricing
KK4711
07959915001
1000 x 20 µL reactions, with fluorescein (Bio-Rad iCycler)
10 mL
Login
for pricing
KK4715
07959931001
5000 x 20 µL reactions (Universal)
50 mL
Login
for pricing
KK4716
07959940001
100 x 20 µL reactions (ROX Low)
1 mL
Login
for pricing
KK4717
07959958001
500 x 20 µL reactions (ROX Low)
5 mL
Login
for pricing
KK4718
07959966001
1000 x 20 µL reactions (ROX Low)
10 mL
Login
for pricing