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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. 

High performing probe-based qPCR Mix for AT-rich, regular and GC-rich templates

Precisely-optimized real time qPCR master mix for probe-based assays. This master mix has been developed for TaqMan® probes but is also suitable for other hydrolysis probe types.

  • suitable for singleplex and duplex assays
  • high specificity and sensitivity
  • one qPCR mix for all cyclers (except capillary)
  • superior results with templates with up to 75% GC content
  • contains dUTP to prevent cross-contamination when used with UNG
  • Benchtop stability for 48 hours for preassambled reactions

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Catalog number

08-17-00001

Quantity

€ 46.50

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Details

Reagents Provided
Item Pcs. Vial size
5x HOT FIREPol® Probe Universal qPCR Mix 1 1 ml | 250 rxn
DMSO 100% 1 0.5 ml
Overview

Description

5x HOT FIREPol® Probe Universal qPCR Mix is optimized for real-time quantitative PCR assays and contains all the components necessary to perform singleplex or duplex qPCR, with the exception of template, primers, and probes. Proprietary reaction buffer that enables efficient amplification of regular and GC-rich targets.

HOT FIREPol® Probe Universal qPCR Mix is optimized for DNA/LNA hydrolysis probes based on the 5' flap endonuclease activity.

The Mix contains internal passive  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.

Applications

  • DNA/LNA hydrolysis probe based assays
  • 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 10 min

Detection type: Probe-based

Reference dye: reference dye based on ROX

Compatible real-time instruments: Most qPCR platforms. Check Your cycler!

Mix Components

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

5x Probe Universal qPCR buffer with 15 mM MgCl2: 1x PCR solution – 3 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® Probe Universal qPCR Mix

Reference dye based on ROX: For multiplex application - if ROX dye is used as one of the fluorophores, internal reference might interfere with the signal.

In separate vial

100% DMSO is included in the kit in a separate vial. DMSO is recommended as a PCR additive for templates with high GC content. In some cases DMSO is also required to relax secondary structures.

qPCR performance in a duplex reaction
Highly competitive qPCR mix
Related Resources

FAQ

Can I use DMSO as a PCR additive in reactions with FIREPol® and HOT FIREPol® DNA polymerases?

Yes, DMSO can be used in reactions with FIREPol® and HOT FIREPol® DNA polymerases and Master Mixes. DMSO is usually recommended for amplifying regions with high GC-content and stable secondary structures. While testing it is recommended to include one sample with additional 2.5% DMSO to test if it improves the results. For further DMSO optimization, the concentration can be raised in 2.5% increments up to 10 % based on following table.

Final DMSO concentration

2.5 %

5 %

7.5 %

10 %

Additional volume of DMSO (in 20 ul PCR reaction)

0.5 µl

1 µl

1.5 µl

2 µl

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.

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.

Storage & Shipping
Storage

Routine storage: -20 oC

Temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of the product

Shipping

At room temperature