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.