KAPA PROBE FAST qPCR Kits

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

KAPA qPCR

Overview
 

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).

Benefits of KAPA PROBE FAST qPCR Kit
  • Fast and reproducible results across several probe chemistries
  • Compatibility with different qPCR protocols

Product highlights

Accurate discrimination between alleles
  • Exceptional differentiation of heterozygous and homozygous alleles*
  • Discrete clusters in SNP genotyping assays


View genotyping data showing discrete allele clusters using KAPA PROBE FAST qPCR Kits.

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

 

View performance data for KAPA PROBE FAST qPCR Kits from multiplex qPCR amplifications of five amplicons.

Increased flexibility
  • Compatible with both standard and fast cycling protocols
  • Retain accurate quantification

 

View data showing comparable qPCR performance with standard or fast protocol using KAPA PROBE FAST qPCR Kits.

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*Data on file.

Specifications

  • Starting Material

    DNA, plasmid DNA, cDNA

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

 

 

   Kit code  2X KAPA PROBE qPCR Master Mix(Universal) 50X KAPA PROBE ROX High 50X KAPA PROBE ROX Low 2X KAPA PROBE qPCR Master Mix (with equipment-specific dye) 2X KAPA PROBE qPCR Master Mix (ROX Low) 10 mM dUTP
Universal KK4701 X X X      
KK4702            
KK4703            
KK4715            
Universal with dUTP KK4721  X X X     X
KK4722             
KK4723            
ABI Prism KK4705       X    
KK4706             
KK4707            
Bio-Rad iCycler KK4709       X    
KK4710            
KK4711            
ROX Low KK4716         X  
KK4717            
KK4718            
KK4719            
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Kits include reagents for RNA fragmentation, cDNA synthesis, and library preparation. Kits with reagents for mRNA capture are also available.

Kit Code Roche Cat. No Description Kit Size
KK4701 07959800001 KAPA PROBE FAST qPCR Kits - 100 x 20 µL reactions (Universal) 1 mL
KK4702 07959818001 KAPA PROBE FAST qPCR Kits - 500 x 20 µL reactions (Universal) 5 mL
KK4703 07959826001 KAPA PROBE FAST qPCR Kits - 1000 x 20 µL reactions (Universal) 10 mL
KK4705 07959842001 KAPA PROBE FAST qPCR Kits - 100 x 20 µL reactions, ROX (ABI Prism) 1 mL
KK4706 07959869001 KAPA PROBE FAST qPCR Kits - 500 x 20 µL reactions, ROX (ABI Prism)5 mL 5 mL
KK4707 07959877001 KAPA PROBE FAST qPCR Kits - 1000 x 20 µL reactions, ROX (ABI Prism) 10 mL
KK4709 07959893001 KAPA PROBE FAST qPCR Kits - 100 x 20 µL reactions, with fluorescein (Bio-Rad iCycler) 1 mL
KK4710 07959907001 KAPA PROBE FAST qPCR Kits - 500 x 20 µL reactions, with fluorescein (Bio-Rad iCycler) 5 mL
KK4711 07959915001 KAPA PROBE FAST qPCR Kits - 1000 x 20 µL reactions, with fluorescein (Bio-Rad iCycler) 10 mL
KK4715 07959931001 KAPA PROBE FAST qPCR Kits - 5000 x 20 µL reactions (Universal) 50 mL
KK4716 07959940001 KAPA PROBE FAST qPCR Kits - 100 x 20 µL reactions (ROX Low) 1 mL
KK4717 07959958001 KAPA PROBE FAST qPCR Kits - 500 x 20 µL reactions (ROX Low) 5 mL
KK4718 07959966001 KAPA PROBE FAST qPCR Kits - 1000 x 20 µL reactions (ROX Low) 10 mL
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What are the recommended applications for KAPA PROBE FAST qPCR Master Mix (2X) Kits?

  • Gene expression analysis
  • SNP genotyping/allelic discrimination
  • Microarray validation
  • NGS validation
     

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 MgCl2concentration 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.

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