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Some applications of this product  may require a license which is not provided by the purchase of this product.

For research use only. 

Highly specific and reproducible dye-based qPCR Mix with blue visualization dye for easy pipetting

Optimized ready-to-use solution for dye-based real-time quantitative
PCR assays. This master mix has been developed to give highly specific and sensitive results, and demonstrates excellent performance with GC-rich regions.

  • high sensitivity with low DNA concentrations
  • blue dye to ease pipetting
  • reduced primer dimer formation
  • one qPCR mix for all cyclers (except capillary)
  • contains dUTP to prevent cross-contamination with UNG treatment

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Catalog number

08-36-00001

Quantity

€ 90.00

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Details

Overview

Description

5x 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 an passive internal reference dye that is compatible with most qPCR cyclers including high ROX or low ROX reference signal requiering 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.

Applications

  • Detection and quantification of DNA and cDNA targets
  • Profiling gene expression
  • Microbial detection
  • Viral load determination

Properties

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!

Mix Components

HOT FIREPol® DNA polymerase: chemically modified FIREPol® DNA Polymerase enabeling hot-start

5x EvaGreen® qPCR buffer with 12. 5 mM MgCl21x 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 5x HOT FIREPol® EvaGreen® Supermix

EvaGreen® dye

Internal reference based on ROX dye

GC-enhancer

Blue visualisation dye

EvaGreen 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!

Trustworthy performance
Related Resources

FAQ

What is the difference between dye-based and probe-based systems?

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.

What is the difference between EvaGreen® and SYBR® Green?

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.
What is the difference between EvaGreen® and SolisGreen® dye?

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.

After first-strand cDNA synthesis, how much cDNA template should I use for my PCR?

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.

Why do certain kits contain a ROX?

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.

What are the storage conditions and stability of Solis BioDyne reagents?

Solis BioDyne products should be stored at -20°C.

Shipping and temporary storage for up to 1 month at room temperature (*1525°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.

Publications

Liver phosphorus content and liver function in states of phosphorus deficiency in transition dairy cows

W. Grünberg et al. / PLoS One / 2019

miR-1933-3p is upregulated in skeletal muscles of MuSK+ EAMG mice and affects Impa1 and Mrpl27

E. Bogatikov et al. / Neurosci. Res. / 2019

Skeletal-muscle-derived mesenchymal stem/stromal cells from patients with osteoarthritis show superior biological properties compared to bone-derived cells

K. Čamernik et al. / Stem Cell Res. / 2019
read more
Storage & Shipping
Storage

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.

Shipping

At room temperature