KAPA SYBR FAST qPCR Kits

Overview

KAPA SYBR FAST qPCR Kits contain the first DNA polymerase engineered via our directed evolution technology to be more tolerant of SYBR Green I dye inhibition. The improved robustness, processivity, and speed of KAPA SYBR FAST qPCR Kits result in consistently high amplification efficiencies enabling more accurate relative quantification for gene expression analysis compared to traditional kits.* 

Benefits of KAPA SYBR FAST qPCR Kits

  • Optimal performance in stringent real-time PCR reaction conditions
  • Improvements to signal-to-noise ratio (fluorescence), quantification cycle (Cq), linearity, and sensitivity compared to other commercial kits*


Refer to Instrument Compatibility Chart for guidance on compatible platforms. 

 

Product Highlights

Quantitate changes in gene expression more accurately

  • High reaction efficiency between 95 – 105%, improving accuracy and reproducibility
  • Unbiased efficiency across a wide range of GC contents and amplicon lengths

Detect low copy and difficult targets consistently

  • Improved processivity results in earlier Cq scores
  • Higher fluorescence detection across varying AT- and GC-rich targets

Complete real-time PCR runs in just 40 minutes

  • 55% shorter run times with fast cycling protocol
  • High performance when switching from standard to fast protocols

*Data on file.

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

Kits include KAPA SYBR FAST qPCR Master Mix (2X), which contains KAPA SYBR FAST DNA Polymerase, reaction buffer, dNTPs, SYBR Green I dye, and MgCl2 at a final concentration of 2.5 mM. Where noted, Master Mixes contain instrument-specific reference dyes, while the Universal kit includes ROX High and ROX Low (both 50X) separately.

Please reference the Instrument Compatibility Chart for guidance on compatible platforms.

Components
   Kit code 2X KAPA SYBR FAST qPCR Master Mix (Universal) 50X KAPA SYBR FAST ROX High
50X KAPA SYBR FAST ROX Low
2X KAPA SYBR FAST qPCR Master Mix (with equipment-specific dye) 2X KAPA SYBR FAST ROX Low Master Mix
Universal

KK4600

KK4601

KK4602

X X X    
ABI Prism

KK4603

KK4604

KK4605

      X  
Bio-Rad iCycler

KK4606

KK4607

KK4608

      X  
Optimized for Roche LightCycler®
480

KK4609

KK4610

KK4611

      X  
ROX Low

KK4619

KK4620

KK4621

KK4622

        X

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Kits include KAPA SYBR FAST qPCR Master Mix (2X), which contains KAPA SYBR FAST DNA Polymerase, reaction buffer, dNTPs, SYBR Green I dye, and MgCl2 at a final concentration of 2.5 mM. Where noted, Master Mixes contain instrument-specific reference dyes, while the Universal kit includes ROX High and ROX Low (both 50X) separately. See Instrument Compatibility Table for more information.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK4600
07959362001
100 x 20 µL reactions (Universal)
1 mL
Contact Us
KK4601
07959389001
500 x 20 µL reactions (Universal)
5 mL
Contact Us
KK4602
07959397001
1000 x 20 µL reactions (Universal)
10 mL
Contact Us
KK4603
07959419001
100 x 20 µL reactions, ROX (ABI Prism)
1 mL
Contact Us
KK4604
07959427001
500 x 20 µL reactions, ROX (ABI Prism)
5 mL
Contact Us
KK4605
07959435001
1000 x 20 µL reactions, ROX (ABI Prism)
10 mL
Contact Us
KK4606
07959443001
100 x 20 µL reactions, with fluorescein (Bio-Rad® iCycler™)
1 mL
Contact Us
KK4607
07959451001
500 x 20 µL reactions, with fluorescein (Bio-Rad iCycler)
5 mL
Contact Us
KK4608
07959460001
1000 x 20 µL reactions, with fluorescein (Bio-Rad iCycler)
10 mL
Contact Us
KK4609
07959478001
100 x 20 µL reactions (optimized for use with Roche® LightCycler 480)
1 mL
Contact Us
KK4610
07959486001
500 x 20 µL reactions (optimized for use with Roche LightCycler 480)
5 mL
Contact Us
KK4611
07959494001
1000 x 20 µL reactions (optimized for use with Roche LightCycler 480)
10 mL Contact Us
KK4617
07959559001
5000 x 20 µL reactions (ABI Prism)
50 mL Contact Us
KK4618
07959567001
5000 x 20 µL reactions (Universal)
50 mL Contact Us
KK4619
07959575001
100 x 20 µL reactions (ROX Low)
1 mL Contact Us
KK4620
07959583001
500 x 20 µL reactions (ROX Low)
5 mL Contact Us
KK4621 07959591001
1000 x 20 µL reactions (ROX Low)
10 mL Contact Us
KK4622
07959605001
5000 x 20 µL reactions (ROX Low)
50 mL Contact Us
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  • Gene expression analysis
  • Microarray validation
  • Low-copy gene detection
  • Gene knockdown validation
  • ChIP
  • Next-generation sequencing library quantification

KAPA SYBR DNA Polymerase is an engineered variant of Taq DNA polymerase. It was engineered specifically for use in SYBR Green-based qPCR through a process of directed evolution. KAPA SYBR DNA Polymerase is both less inhibited by SYBR Green I dye and more processive than wild-type Taq DNA Polymerase, resulting in increased sensitivity, fluorescence and speed. The enzyme has an integrated antibody-mediated hotstart so that it remains inactive during reaction set-up, minimizing the formation of non-specific products.

  • Reduced inhibition to SYBR Green I dye and hence higher PCR efficiencies at elevated SYBR Green I concentrations.
  • The increased activity of KAPA SYBR DNA Polymerase provides the ability to formulate stringent buffering conditions suitable for amplicons that are difficult to amplify, due to either high AT or high GC content. Wild-type Taq DNA polymerase does not function in this proprietary buffer.
  • Shorter overall run times due to increased processivity of the engineered enzyme over kits containing wild-type Taq DNA polymerases.

For optimal quantitative results, use up to 20 ng of genomic DNA or plasmid DNA per 20 µL reaction. For two-step RT-PCR, use either undiluted or diluted cDNA generated from up to 1 µg of total RNA. The volume of the cDNA (reverse-transcription product) should not exceed 10% of the final PCR volume (e.g. for a 20 µL qPCR reaction, use up to 2 µL of undiluted cDNA).

 

For certain real-time cyclers, ROX passive reference dye is used to compensate for non-PCR-related variations in fluorescence detection. Fluorescence from the ROX reference dye does not change during the course of qPCR, but provides a stable baseline against which PCR-related fluorescent signals are normalized. Thus, ROX compensates for differences in fluorescence detection between wells due to slight variations in reaction volume or differences in well position. The use of ROX (either ROX High or ROX Low) is necessary for all instruments from Applied Biosystems and is optional for the Stratagene Mx series instruments. Instruments from Bio-Rad/MJ Research, Cepheid, Corbett Research, Qiagen, Eppendorf and Roche do not require ROX (although it is important to note that the Bio-Rad iCyclers require fluorescein as a passive reference dye). The presence of ROX in the Master Mix does not interfere with qPCR on any instrument, since the dye is not involved in the reaction and has an emission spectrum different from that of SYBR Green I.

The KAPA SYBR qPCR Master Mix(2X) providesMgCl2 at an optimized final concentration of 2.5 mM. It is highly unlikely that additional MgCl2 will improve reaction efficiency or specificity.

Although the antibody-mediated hot-start KAPA SYBR DNA Polymerase is activated after 10 seconds at 95°C, optimal denaturation of template may require up to 3 minutes. Human genomic DNA, for example, or templates with a high GC content, would require a longer initial denaturation time than plasmid DNA.

One of the most critical factors in maximizing amplification efficiency during SYBR Green-based qPCR is optimal primer annealing and extension. Optimal primer annealing times depend on the sequence and length of the primers. This means that the minimum annealing times can possibly be reduced from 20 seconds to 15 seconds, but this has to be determined empirically. Due to the extremely high processivity of KAPA SYBR DNA Polymerase, an extension time of 1 second for amplicons up to 300 bp is sufficient, should a 3-step protocol be used.

When optimal primer-annealing temperatures of primers are lower than 60ºC, it is advisable to convert to a 3-step protocol. For example, if the optimal annealing temperature is 55ºC, then perform annealing for 20 seconds at 55ºC, followed by a 1 second extension and data acquisition at 72ºC.

 

The reasons for primer dimer formation in an NTC are often due to multiple factors. These include: sub-optimal primer annealing temperature (often due to differences in buffering conditions between different qPCR kits), sub-optimal primer synthesis (HPLC-purified primers result in less primer dimer formation and are useful for low copy number detection), and poor primer design (using software such as Primer3 is recommended when designing primers). An alternative is to reduce the total number of cycles in a qPCR reaction if amplification of the primer dimer lies outside of the range of the experimental data eg. if the sample being tested has an average Cq of 28 cycles and contains specific product only (no primer dimer), and the NTC amplifies a primer dimer after 37 cycles, the reaction can be run for 35 cycles, rather than 40. This approach can be taken only when the sample being tested gives rise to a specific product and not a combination of specific product and primer dimer, and also when the target is not likely to present in low copy numbers.

 

The reason for the KAPA SYBR FAST qPCR Kit’s outperformance of other kits on the market is primarily a result of its superior processivity and the robustness of the KAPA SYBR DNA Polymerase over kits containing wild-type Taq DNA Polymerase. There will be situations, particularly with targets that are easy to amplify, where the performance of the KAPA SYBR FAST qPCR Kit will be similar to competitor kits. Generally, the most significant differences in performance will be noticed when amplifying from complex targets such as human genomic DNA or when amplifying templates with either high AT or GC content. It is important to note that another reason for poor performance against another kit would be if the cycling conditions for the KAPA SYBR FAST qPCR Kit are not followed—using cycling designed for a wild-type Taq DNA Polymerase (often with a chemically-mediated hot start) will severely compromise the efficiency of our engineered polymerase.

The correct concentration and amount of reference dye added to the KAPA SYBR FAST qPCR Master Mix is critical for optimal analysis. If the incorrect concentration or amount of reference dye is added to the Master Mix, the normalized signal may be lower than expected (if too much ROX has been added), or higher than expected (if too little ROX has been added). Always thaw and mix solutions before use. Consult the relevant KAPA SYBR FAST qPCR Kit User Guide in order to determine the correct amount and concentration of ROX to be added. If using ABI instrumentation, analysis of the raw signal can also be performed with the ROX filter switched off.

The KAPA SYBR FAST qPCR Master Mix is stable at 4ºC although it is also possible to store the kit at -20ºC. The kit is sensitive to light and should be protected from direct light, particularly during storage. Care should also be taken to avoid freeze-thaw cycles. When stored under these conditions and handled correctly, full activity of the kit is retained for at least 12 months, as is indicated on the kit label.

 

Ensure that the KAPA SYBR FAST qPCR Master Mix contained ROX High (if using the Universal Kit). If no ROX was added to the Master Mix, or the incorrect ROX was added, rerun the analysis with the ROX filter switched off.

Yes. However, the Roche LightCycler 1.2, 1.5 and 2.0 capillary instruments require the addition of unacetylated 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.

High reaction efficiency combined with optimal primer design and primer purification is critical in achieving single copy detection. Reaction efficiency can be optimized using an annealing temperature gradient. A suitable qPCR primer design program should be used when designing primers (e.g. Primer3) and HPLC purification of primers is strongly recommended in order to reduce the formation of PCR artifacts.