HOT FIREPol^® EvaGreen^® qPCR Mix Plus

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.

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:

• EvaGreen^® dye has less background than SYBR^® Green I due to its novel “release-on-demand” DNA-binding mechanism,
• EvaGreen^® is less inhibitory in the PCR reaction than SYBR^® Green I, enabling the use of higher dye concentration for maximal signal and better high-resolution DNA melt analysis,
• EvaGreen^® is non-mutagenic, non-cytotoxic and environmentally safe,
• EvaGreen^® is very stable under PCR condition and during routine storage and handling, whereas SYBR^® Green I is known to degrade following multiple freeze-thaw cycles and under PCR conditions.

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.