Precise, reproducible, and versatile for all probe-based qPCR applications.
KAPA PROBE FAST qPCR 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).
Optimized for versatility and speed - KAPA PROBE FAST qPCR Kits provide fast and reproducible results for genotyping, gene expression analysis, and multiplexing offering:
- Compatibility with all probe-based qPCR applications and instruments
- Fast, reproducible, and precise quantification
- Discrete clusters in SNP genotyping assays
- Multiplex qPCR
- Broad dynamic range
- Highly stable master mix for high-throughput workflows
Application Note: Fast Real-Time PCR
Kapa Biosystems and Eppendorf North America released a new application note on the use of KAPA PROBE FAST qPCR Kits and the Eppendorf Mastercycler® ep realplex for high-throughput fast qPCR.
KAPA PROBE FAST qPCR Kits are designed for high throughput, fast-cycling, real-time PCR using sequence-specific fluorogenic probes. These kits are compatible with all fluorogenic probe-based technologies, including hydrolysis probes (e.g. TaqMan®) and displacement probes (e.g. molecular beacons).
The KAPA PROBE FAST qPCR Master Mix (2X) is a ready-to-use cocktail containing all components except primers, probe(s) and template for fast cycling probe-based real-time PCR. The 2X Master Mix contains KAPA Taq HotStart DNA polymerase, KAPA PROBE FAST qPCR Buffer, dNTPs, MgCl2 and stabilizers.
KAPA Taq HotStart DNA Polymerase is an antibody-mediated hot start formulation of KAPA Taq DNA polymerase. In the HotStart formulation, the enzyme is combined with a proprietary antibody that inactivates the enzyme until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency.
Discrete clusters and high call rates for accurate and reproducible allelic discrimination
All 168 human genomic DNA samples were accurately genotyped using the ABI sequence detection system (SDS) version 2.3 software (autocaller confidence level 95%). 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. Reactions were performed in 5 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA (10 ng per reaction), 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: 95 ºC for 10 min followed by 40 cycles of 95 ºC, 15 sec; 60 ºC, 60 sec.
Fast, high performance five-color multiplexed qPCR
Highly reproducible and efficient results for all 5 amplicons across a 5 point dilution series of human genomic DNA were obtained when assayed in penta-plex using a fast cycling protocol. Standard curves were generated using 4-fold dilutions of human genomic DNA (0.39 – 100 ng per reaction) tested in triplicate using the Corbett Rotor-Gene™ 6000 HRM real-time rotary analyzer. Reactions were performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA, 200 nM of each primer and 200 nM of each hydrolysis probe (ACTB - FAM™/BHQ®-1, ERBB2 - CAL Fluor® Gold 540/ BHQ®-1, ERBB3 - CAL Fluor® Orange 560/ BHQ®-2, EGFR - CAL Fluor® Red 610/ BHQ®-2, ACTG1 - Quasar® 705/ BHQ®-2) using the following fast cycling protocol: 95 ºC for 3 min followed by 40 cycles of 95 ºC, 15 sec; 60 ºC, 15 sec.
Precise and highly reproducible discrimination
Excellent reproducibility and discrimination across samples of low copy number with similar abundance levels was achieved. Reactions were performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA (1.5-fold dilutions over a 89 - 450 copies per reaction range), 200 nM of each primer and 200 nM of hApoB100 (FAM/ BHQ®-1) hydrolysis probe using a fast cycling protocol (95 ºC for 3 min followed by 40 cycles of 95 ºC, 3 sec; 60 ºC, 20 sec) on the Corbett Rotor-Gene ™ 6000 HRM real-time rotary analyzer.
Broad linear dynamic range of up to 10 orders of magnitude using a fast cycling protocol
Highly reproducible and 100% efficient qPCR was achieved across the 10 log-fold dilution series. Amplification plots were generated using 10-fold dilutions of a synthetic oligonucleotide target (60 – 6 x 106 copies per reaction) tested in triplicate using the Corbett Rotor-Gene ™ 6000 HRM real-time rotary analyzer. Reactions were performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, synthetic DNA, 200 nM of each primer and 200 nM of hApoB100 (FAM/ BHQ®-1) hydrolysis probe using the following fast cycling protocol: 95 ºC for 3 min followed by 40 cycles of 95ºC, 3 sec; 60 ºC, 30 sec.
High performance with both standard and fast cycling protocols
Excellent reproducibility and efficiency was achieved using both cycling protocols. Reactions were performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA (10-fold dilutions over a 0.1 - 100 ng per reaction range), 200 nM of each primer and 200 nM of hApoB100 (FAM/ BHQ®-1) hydrolysis probe using either a standard cycling protocol (95 ºC for 10 min followed by 40 cycles of 95ºC, 15 sec; 60 ºC, 60 sec) or a fast cycling protocol (95 ºC for 3 min followed by 40 cycles of 95 ºC, 3 sec; 60 ºC, 20 sec) on the Corbett Rotor-Gene ™ 6000 HRM real-time rotary analyzer.
Excellent benchtop stability
KAPA PROBE FAST qPCR Master Mix offers the flexibility to perform experiments over multiple days. Reactions were left on the bench and in the dark for 0, 24, 48, 72, and 96 hours after assembly. Real-time PCR was performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA (10-fold dilutions over a 0.1 - 100 ng per reaction range), 200 nM of each primer and 200 nM of hApoB100 (FAM/ BHQ®-1) hydrolysis probe using the following fast cycling protocol: 95 ºC for 3 min followed by 40 cycles of 95 ºC, 3 sec; 60 ºC, 30 sec. The data represents the average of 3 replicates for each DNA dilution and time point.
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.
A license to perform the patented 5’ Nuclease Process for research is obtained by the purchase of (i) both Authorized 5' Nuclease Core Kit and Licensed Probe, (ii) a Licensed 5’ Nuclease Kit, or (iii) license rights from Applied Biosystems.
This product is an Authorized 5’ Nuclease Core Kit. Use of this product is covered by one or more of the following claims outside the US corresponding to US Patent No. 5,210,015, 5,487,972, 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. Separate purchase of a Licensed Probe would convey rights under US Patents and corresponding patent claims outside the US: 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569, 5,804,375 (claims 1-12 only), and and claims outside the United States corresponding to US Patent No. 6,214,979. No right under any other patent claim 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 from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Licensed under U.S. Patent nos. 5,338,671 and 5,587,287 and corresponding patents in other countries.
iCycler®, Mx3000P®, Mx3005™, Mx4000®, Rotor-Gene™, DNA Engine Opticon™, Chromo 4™, LightCycler® , Smart Cycler®, ROX and TaqMan® are trademarks or registered trademarks of their respective companies.
Frequently Asked Questions
1. What is the enzyme in KAPA PROBE FAST qPCR Master Mix (2X) kits?
This product contains KAPATaq HotStart DNA Polymerase. It is based on the single-subunit, wild-type Taq DNA polymerase of the thermophilic bacterium Thermus aquaticus. In the HotStart formulation, the enzyme is combined with a proprietary antibody that inactivates the enzyme until the first denaturation step.
2. 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 95ºC, optimal denaturation of template may require up to 3 minutes.
3. 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).
4. 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 -20 ºC or -80 ºC 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 -20 ºC or -80 ºC. To ensure optimum activity, fluorescent probes should always be protected from light to avoid photobleaching. Probes and primers stored at -20 ºC or -80 ºC are stable for over one year.
5. At what concentration should I use my probes in qPCR?
Probes are generally used at a final concentration of 100 nM – 400 nM. We suggest starting with 200nM final concentration.
6. At what concentration should I use my primers in qPCR?
Primers are generally used at a final concentration of 100 nM – 400 nM. We suggest starting with 200nM final concentration.
7. 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.
8. 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. Clean working 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.
9. How should I store KAPA PROBE FAST qPCR kits?
KAPA PROBE FAST qPCR kits should be stored at -20 ºC for long term storage up to 1 year from receiving the kit. This kit retains stability and performance up to 30 freeze thaw cycles. For short term storage it may be more convenient to store the kit at 4 ºC for up to 3 months. Always protect the kit from light if it contains either Rox or Fluorescein reference dyes.
10. With which probe chemistries can KAPA PROBE FAST qPCR Master Mix kits be used?
KAPA PROBE FAST 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.
11. 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.
12. When do I use Rox passive reference dye?
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.