What are the recommended applications for KAPA SYBR FAST qPCR Master Mix (2X) Kits?
- Gene expression analysis
- Microarray validation
- Low-copy gene detection
- Gene knockdown validation
- Next-generation sequencing library quantification
What is the enzyme in the KAPA SYBR FAST qPCR Master Mix (2X) Kits?
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.
What are the benefits of using KAPA SYBR DNA Polymerase for SYBRGreen I-based qPCR?
- 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.
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 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).
When would I add ROX passive reference dye to my qPCR reaction?
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.
Do I need to add additional magnesium chloride to my qPCR reaction?
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.
Why would I require initial activation times of greater than 10 sec at 95ºC for an antibody-mediated hotstart DNA polymerase?
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.
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. 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.
Why would I use a 3-step cycling protocol rather than a 2-step protocol?
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.
How do I eliminate primer dimer formation in the no template control (NTC)?
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.
Why does the KAPA SYBR FAST qPCR Kit not outperform competitor kits in all situations?
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.
Why is the signal intensity of the KAPA SYBR FAST qPCR Master Mix (2X) Kit not as high as expected after ROX normalization?
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.
What are the storage recommendations for KAPA SYBR FAST qPCR Kits?
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.
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 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.
Can KAPA SYBR FAST qPCR Kits be used with the Roche LightCycler 2.0 capillary qPCR instrument?
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.
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 when designing primers (e.g. Primer3) and HPLC purification of primers is strongly recommended in order to reduce the formation of PCR artifacts.