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For research use only.
Optimized ready-to-use solution for dye-based real-time quantitativePCR assays. This master mix has been developed to give highly specific and sensitive results and demonstrates excellent performance with GC-rich regions.
HOT FIREPol® EvaGreen® qPCR Supermix is an optimized 5X reaction mix for dye-based qPCR detection and quantitation of target DNA sequences using EvaGreen® dye and SYBR®/FAM channel of most real-time qPCR instruments.
The Mix contains HOT FIREPol® DNA Polymerase and a passive internal reference dye that is compatible with most qPCR cyclers including no ROX and low ROX* reference signal requiring platforms.
The Mix contains dUTPs to prevent cross-contamination with UNG treatment and visible dye to ease reaction set-up. This dye does not interfere with real-time detection.
Concentration: 5x
Hot-start: yes, initial activation in 12 min
Detection type: dye-based, includes EvaGreen® intercalating dye
Reference dye*: based on ROX
Compatible real-time instruments: most qPCR cyclers. Check Your cycler!
IMPORTANT UPDATE! HOT FIREPol® EvaGreen® qPCR Supermix is not compatible with high ROX cyclers such as Applied BioSystems® StepOne™ or StepOnePlus™
HOT FIREPol® DNA polymerase: chemically modified FIREPol® DNA Polymerase enabling hot-start
5x EvaGreen® qPCR buffer with 12. 5 mM MgCl2: 1x PCR solution – 2.5 mM MgCl2
dNTPs, including dUTP: mix allows UNG treatment to prevent carryover contamination from previous runs.IMPORTANT: UNG is not included in the HOT FIREPol® EvaGreen® Supermix
EvaGreen® dye
Internal reference based on ROX dye
GC-enhancer
Blue visualisation dye
EvaGreen® is a DNA-binding dye with many features that make it a superior alternative to SYBR® Green I for qPCR. Apart from having similar spectra, EvaGreen® has three important features that set it apart from SYBR® Green I: EvaGreen® has much less PCR inhibition, is an extremely stable dye, and has been shown to be non-mutagenic and non-cytotoxic. EvaGreen® is compatible with all common real-time PCR cyclers – simply select the standard settings for SYBR® Green or FAM!
Dye-based detection (e.g., EvaGreen®, SolisGreen®, SYBR® Green) is a cost-effective qPCR option, as it requires only addition of PCR primers. However, the intercalating dye will detect any dsDNA (non-specific amplicons, primer dimers) produced in the reaction. Melt curve analysis performed after the qPCR can be used to verify the specificity of amplification and to check for the presence of non-specific amplification products.
The probe-based qPCR system demonstrates higher specificity compared to dye-based qPCR, because probes only detect the gene of interest. Keep in mind that in probe-based assays, primer dimers and non-specific products will not be detected, however, they may compromise the PCR efficiency. Using probe-based qPCR system, it is possible to distinguish between sequences with high similarity (e.g. single-nucleotide variations). Additionally, probe-based qPCR assays allow for multiplex reactions in one tube, while only a single target can be amplified and measured in a dye-based qPCR.
Both dyes are DNA intercalating agents that are used to stain dsDNA.
EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means that no change in optical settings is required for using an EvaGreen®-based qPCR mix.
EvaGreen® was reported to have several benefits over SYBR® Green I:
Both dyes are DNA intercalating agents that are used to stain dsDNA. SolisGreen™ dye is spectrally similar to EvaGreen® allowing detection in the FAM or SYBR® Green I channel (SolisGreen™ excitation maximum 489 nm, emission maximum 515 nm). It has been shown that qPCR mixes based on SolisGreen™ dye have increased performance with low target concentration demonstrating improved precision and less variance between technical replicates.
Recommended final amount of cDNA sample in downstream PCR reaction is up to one tenth of the final reaction volume. Overload of cDNA sample may compromise the downstream PCR, because cDNA sample may contain reaction components that may inhibit your PCR reaction.
ROX (carboxy-X-rhodamine) is one of the passive reference dyes providing a constant fluorescent signal that is used for signal normalization during the amplification cycles. The emission recorded from a reference dye during the baseline cycles is used to normalize the emission recorded from the reporter (e.g. EvaGreen®, SolisGreen®, SYBR® Green, etc.) in later cycles in ROX-dependent real-time PCR systems (e.g. Applied Biosystems 5700, 7000, 7300, 7700, 7900HT, StepOne™, StepOnePlus™). Reference dye compensates for small fluorescent fluctuations and well-to-well variations that may occur.
In Solis BioDyne qPCR mixes we use technology, based on ROX, that allows us to use same mix for high- and low- ROX requiering cyclers.
Please check cycler-mix compatibility here if not sure.
Solis BioDyne products should be stored at -20°C.
Shipping and temporary storage for up to 1 month at room temperature (*15−25°C) has no detrimental effects on the quality of Solis BioDyne reagents.
Freeze-thaw stability is tested for each product. Most PCR and qPCR products have passed 30 freeze-thaw cycles with no changes in performance. Specific information is found in Storage and Shipping conditions of each product.
When stored and handled under the recommended conditions, full activity of the reagents is retained until the Expiry Date printed on the tube label.
*World Health Organization (2003). Guidelines for the Storage of Essential Medicines and Other Health Commodities.
Routine storage: -20oC
Temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of HOT FIREPol® EvaGreen® qPCR Supermix.
At room temperature
Ready-to -use solution for simple dye-based qPCR assays, incorporating EvaGreen® dye. Available for ROX, no ROX, and capillary cyclers.
Highly sensitive dye-based qPCR Mix with improved detection of low-copy targets. Suitable for ROX-dependent and ROX-independent qPCR cyclers.
Extremely fast and highly sensitive dye-based qPCR that includes novel in-silico designed SolisFAST® DNA Polymerase Mix for most demanding applications
High-performing probe-based qPCR Mix for AT-rich, regular and GC-rich templates.
All-inclusive and the most convenient product format for first-strand cDNA synthesis. A combined enzyme mix (FIREScript® and RiboGrip®) and a reaction premix in separate tubes allowing you to choose the most appropriate priming option.
