KAPA SYBR® FAST qPCR Kits
Containing the first DNA polymerase evolved for real-time PCR.
KAPA SYBR® FAST qPCR Kits are designed for the highest performance in real-time PCR. Kits contain a novel DNA polymerase engineered through a process of molecular evolution - the result is a unique enzyme, specifically evolved for qPCR using SYBR® Green I dye chemistry.
The KAPA SYBR® DNA Polymerase was engineered to perform optimally in stringent real-time qPCR reaction conditions, exhibiting dramatic improvements to signal-to-noise ratio (fluorescence), cycle threshold (Ct), linearity, and sensitivity. The KAPA SYBR® DNA Polymerase and proprietary buffer system enhances the amplification efficiency of difficult templates, including both GC-rich and AT-rich templates.
I lead genome-wide studies on RNA (gene expression and alternative splicing) and DNA (SNPs and copy number variation) and real-time PCR is a key player for validating our results. I switched to the KAPA SYBR® FAST qPCR Kit from Kapa Biosystems because I noticed a significant improvement in PCR reaction efficiency - the kit works very nicely for the vast majority of primers we design without optimization and for all our various applications. This universality makes our real-time PCR assays very fast to establish and our results more reliable.
- Dr. Philippe Demouging, Department of Molecular Psychology, University of Basel, Switzerland
Application Note: Fast Real-Time PCR
Kapa Biosystems and Eppendorf North America released a new application note on the use of KAPA SYBR® FAST qPCR Kits and the Eppendorf Mastercycler® ep realplex for high-throughput fast qPCR. Download here>>
Application Note: Gene Expression Analysis
KAPA SYBR® FAST qPCR Kits are formulated for the highest performance in gene expression analysis. For more information on the use of KAPA SYBR® FAST for gene expression download the new application notes.
KAPA SYBR® FAST qPCR Kits are supplied as a ready-to-use cocktail containing all components except primers and template, for the amplification and detection of DNA in qPCR.
The KAPA SYBR® FAST qPCR Kit is supplied as a 2x Master Mix with integrated antibody-mediated hot start, SYBR® Green I fluorescent dye, MgCl2, dNTPs, and stabilizers. ROX reference dye is included in the ABI Prism® Master Mix (2X), and supplied in separate tubes in the Universal Master Mix (2X). Fluorescein reference dye is included in the Bio-Rad iCycler Master Mix (2x). The KAPA SYBR® FAST qPCR Master Mix (2X) optimized for the Roche LightCycler® 480 does not contain reference dye.
KAPA SYBR® FAST qPCR Kits are intended for use on all real-time instruments and protocols.
KAPA SYBR® FAST qPCR Kits are ideally suited for:
- Gene expression analysis
- Low copy gene detection
- Microarray validation
- Gene knockdown validation
- High-throughput qPCR
- Next-generation DNA sequencing library quantification
High performance gene expression analysis
The improved speed, processivity and robustness of KAPA SYBR® FAST qPCR Kits results in consistently high amplification efficiencies required for accurate relative quantification. To demonstrate the high performance of KAPA SYBR® FAST for gene expression analysis, the reaction efficiencies obtained for ten commonly used housekeeping genes in the human breast cancer cell line, MCF-7, were compared. The KAPA SYBR® FAST qPCR Kit achieved consistently high amplification efficiencies (95 – 104%) across all ten genes, despite differences in amplicon length.
No bias in amplification efficiency relative to GC content (44.2 – 62.5%) or amplicon length (86 – 249 bp) is observed with KAPA SYBR® FAST. Ampliﬁcation efficiencies achieved for ten housekeeping genes with the KAPA SYBR® FAST Universal qPCR Kit, plotted against GC content (top) or amplicon length (bottom). The reaction efficiency achieved for each of the ten genes fell within the optimal range of 95 –105%, independent of the nature or length of the amplicon.
Typical results achieved with KAPA SYBR® FAST qPCR Kits in the ampliﬁcation of housekeeping genes for gene expression analysis. Two housekeeping genes (ActB and HPRT1) were ampliﬁed from log-fold serial dilutions of MCF-7 cDNA (100 ng - 10 pg/reaction) using the KAPA SYBR® FAST Universal qPCR Kit (green) or the Invitrogen Express SYBR® GreenER™ Kit (blue). Linear ampliﬁcation plots (top) demonstrate earlier Ct scores and greater baseline subtracted ﬂuorescence for both the ActB and HPRT1 genes with the KAPA SYBR® FAST Kit. Calculated reaction eﬃciencies (bottom) conﬁrmed that the consistently high performance required for accurate expression quantitation is achievable with the KAPA SYBR® FAST qPCR Kit (EActB = 100% and EHPRT1 = 104%). Eﬃciencies obtained with the Invitrogen Express SYBR® GreenER™ Kit were sub-optimal for both housekeeping genes (EActB= 107% and EHPRT1= 115%).
Superior signals, Ct values, and reaction efficiencies
The high performance of the KAPA SYBR® FAST qPCR kit can been seen when compared to other leading Fast qPCR Mater Mixes. KAPA SYBR® FAST consistently produces higher signal, earlier Cts, and 100% reaction efficiencies.
A 290 bp fragment of the human beta-actin gene was amplified from a set of five 10-fold dilutions of human genomic DNA (20 ng to 2 pg) using KAPA SYBR® FAST qPCR Master Mix (green) or fast qPCR competitor kits (ABI Power SYBR® Green qPCR Master Mix, orange; Qiagen QuantiFast qPCR Master Mix, purple; and Invitrogen SYBR® GreenER™ qPCR SuperMix, blue). Reactions were performed according to each competitors’ suggested protocol on an ABI Prism® 7900 HT real-time cycler.
High speed and high performance
High speed does not compromise performance with the KAPA SYBR® FAST qPCR kits. As can been seen from the amplification plots and standard curves there is no loss in performance between fast and conventional cycling conditions even with complex targets.
Standard Cycling Fast Cycling Activation: 5 min @ 95°C 3 min @ 95°C PCR Cycling: 15 s @ 95°C 1 sec @ 95°C 60s @ 60°C 20s @ 60°C Cycle Time: 40 cycles = 55 min 40 cycles = 14 min Elapsed Time: 1hr. 30 min 40 min RotorGene 6000HRM 5 runs/ 8hr shift 12 runs/8hr shift
Human coagulation factor V (F5) was amplified from log-fold dilutions of human genomic DNA (20 ng-2 pg) using KAPA SYBR® FAST qPCR Master Mix, with fast cycling protocol, or with conventional cycling conditions. There is no loss in performance between fast and conventional cycling conditions even with complex targets. Although the proprietary antibody hot start is activated after 20 seconds at 95°C, 3 min initial activation at 95°C is recommended for optimal denaturation of complex targets.
Highest performance on Roche LightCycler® 480
KAPA SYBR® FAST qPCR Master Mix (2x) is specifically formulated for the highest performance on the Roche LightCycler® 480 instrument. A dramatic loss in performance of the Roche LightCycler® 480 SYBR® Green I Master kit was observed as the extension time at 72°C was reduced from 6 sec to 1 sec. There was no loss in performance of the KAPA SYBR® FAST qPCR Master Mix (2x) Kit optimized for the Roche LightCycler® 480.
Human B-actin (290 bp) was amplified from log-fold dilutions of human genomic DNA (20 ng to 2 pg) using KAPA SYBR® FAST qPCR Master Mix (2x) Roche LightCycler® 480 or Roche LightCycler® 480 SYBR Green I Master kit (Roche). Comparisons were performed according to the competitor protocol A: Roche protocol: 95°C, 5 min followed by 40 cycles of 95°C, 10 sec; 60°C, 15 sec; 72°C, 6 sec; or B: KAPA SYBR® FAST Protocol: 95°C 3 min followed by 40 cycles of 95°C, 10 sec; 60°C, 15 sec; 72°C, 1 sec.
Highest fluorescence and earliest Ct
The KAPA SYBR® FAST qPCR kit displays the highest fluorescence and earliest Ct values from a range of AT and GC-rich amplicons.
Human B-actin, human coagulation factor V (F5), and HPRT1 targets were amplified from 20ng (5900 copies) of human genomic DNA on an ABI Prism® 7900 HT sequence detection system. Values represent an average of triplicates.
Certain applications of this product are covered by patents issued to parties other than Kapa Biosystems and applicable in certain countries. Purchase of this product does not include a license to perform any such applications. Users of this product may therefore be required to obtain a patent license depending upon the particular application and country in which the product is used.
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056, 6,171,785, and 5,928,907 (claim numbers 12-24, 27-28). The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
This product is provided under an agreement between Molecular Probes, Inc. and Kapa Biosystems Inc., and the manufacture, use, sale or import of this product is subject to one or more of U.S. Patent Nos. 5,436,134; 5,658,751 and corresponding international equivalents, owned by Molecular Probes, Inc. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851.
Licensed under U.S. Patent nos. 5,338,671 and 5,587,287 and corresponding patents in other countries.
The purchase of this product includes a limited, non-transferable license under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein.
SYBR® is a registered trademark of Molecular Probes, Inc, Oregon. PRISM® and GeneAmp® are registered trademarks of Applera Corporation. iCycler®, Mx3000P®, Mx3005™, Mx4000®, Roto-Gene™, DNA Engine Opticon™, Chromo 4™, LightCycler® , and Smart Cycler® are trademarks or registered trademarks of their respective companies.
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1. What are the recommended applications for KAPA SYBR qPCR Master Mix (2X) Kits?
- Gene expression analysis
- Microarray validation
- Low-copy gene detection
- Gene knockdown validation
- Next-generation sequencing library quantification
2. What is the enzyme in KAPA SYBR® qPCR Master Mix (2X) kits?
KAPA SYBR® DNA Polymerase is an engineered variant of Taq DNA polymerase. It was specifically engineered for use in SYBR® Green- based qPCR through a process of molecular evolution. KAPA SYBR® DNA Polymerase is less inhibited by SYBR® Green I dye and is more processive than wild-type Taq DNA Polymerase resulting in increased sensitivity, fluorescence and speed. The enzyme has an integrated antibody-mediated hot-start to remain inactive during reaction set-up, minimizing the formation of non-specific products.
3. What are the benefits of using KAPA SYBR® DNA Polymerase for SYBR® Green I-based qPCR?
- Reduced inhibition to SYBR® Green I dye and hence higher PCR efficiencies at elevated SYBR® Green I concentrations.
- The ability to formulate stringent buffering conditions suitable for difficult to amplify amplicons due to either high AT or high GC content. This is due to the increased activity of KAPA SYBR® DNA Polymerase. 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.
4. How much template should I add to my qPCR reaction?
For optimal quantitative results, use up to 20 ng of genomic DNA or plasmid DNA per 20 µl reaction (for smaller volumes, the amount of template should be decreased proportionally). 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 2.0 µl of undiluted cDNA).
5. 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. The presence of ROX dye in the master mix does not interfere with real-time PCR on any instrument, since the dye is not involved in the reaction and has an emission spectrum different from that of SYBR® Green I.
6. Do I need to add additional magnesium chloride to my qPCR reaction?
The KAPA SYBR® qPCR Master Mix (2X) provides MgCl2 at an optimized final concentration of 2.5 mM. It is highly unlikely that additional MgCl2 will improve reaction efficiency or specificity.
7. 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 KAPA SYBR® DNA Polymerase is activated after 10 seconds at 95 ºC, 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.
8. What is the shortest possible combined extension/annealing time?
One of the most critical factors in maximizing amplification efficiency during SYBR® Green-based qPCR is optimal primer annealing and extension between cycles. Optimal primer annealing times depend on the sequence and length of the primers and hence the minimum annealing times can be reduced from 20 sec to 15 sec but this has to be determined empirically. Due to the extremely high processivity of the KAPA SYBR® DNA Polymerase, an extension time of 1 sec for amplicons up to 300 bp is sufficient.
9. Why would I use a 3 step cycling protocol rather than a 2 step protocol?
When optimal primer annealing temperatures of primers are less than 60 ºC, it is advisable to convert to a 3 step protocol. If the optimal annealing temperature is 55 ºC, then perform annealing for 20 sec at 55 ºC followed by 1 sec extension and data acquisition at 72 ºC.
10. How do I eliminate primer dimer in the no template control (NTC)?
The reasons for primer dimer formation in a NTC control 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 in primer design). Reducing the total number of cycles in a qPCR reaction is an alternative method if amplification of the primer dimer lies outside of the range of the experimental data. For example, if the sample being interrogated has a Ct 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 specific product and not a combination of specific product and primer dimer.
11. Why does the KAPA SYBR® FAST kit not outperform competitor kits in all situations?
The outperformance of the KAPA SYBR® FAST kit is based primarily on superior processivity and robustness of the KAPA SYBR® DNA Polymerase over kits containing wild-type Taq DNA Polymerase. There will be situations, particularly with easy to amplify targets, where the performance of KAPA SYBR® FAST 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.
12. Why is the signal intensity of the KAPA SYBR® FAST qPCR Master Mix (2X) Universal Kit not as high as expected after ROX normalization?
The correct concentration and amount of reference dye added to the KAPA SYBR® FAST master mix is critical for optimal instrumentation 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 technical data sheet (TDS) for determining the correct amount and concentration of ROX to add. If using ABI instrumentation, analysis of the raw signal can always be performed with the ROX filter switched off.
13. What are the storage recommendations for KAPA SYBR® FAST qPCR kits?
The KAPA SYBR® FAST qPCR Master Mix is stable at 4 ºC although it is also possible to store the kit at -20 ºC. The kit is sensitive to light and should be protected from direct light particularly during storage. Care should 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 indicated on the kit label.
14. I am using an ABI 7900 HT instrument and after cycling there is no signal in the amplification window but specific product is present when run on an agarose gel.
Ensure that the KAPA SYBR® FAST master mix contained ROX (if using the Universal Kit). If no ROX was added to the master mix, rerun the analysis with the ROX filter switched off.
15. Can KAPA SYBR® 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.
16. What is the best way to achieve single copy detection?
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 in designing primers (e.g., Primer3). HPLC purification of primers is strongly recommended to reduce the formation of primer dimers.