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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. 

Sensitive qPCR Mix designed for effective quantification of DNA targets with probe-based chemistry

Cost-effective real-time qPCR master mix for probe-based qPCR assays.
This master mix has been developed for TaqMan® probes but is suitable for other hydrolysis probe types as well.

  • suitable for singleplex and duplex assays
  • high specificity and sensitivity
  • reaction set-up and shipment without dry ice

Ordering

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Catalog number

08-14-00001

Quantity

€ 75.00

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Details

Overview

Description

HOT FIREPol® Probe qPCR Mix Plus (ROX) is optimized for real-time quantitative PCR assays and contains all the components necessary to perform qPCR, with the exception of template, primers, and probe.

The qPCR Mix contains optimized components and HOT FIREPol® DNA Polymerase supplied in a proprietary reaction buffer that enables detection of low copy number targets. HOT FIREPol® Probe qPCR Mix Plus (ROX) is optimized for DNA hydrolysis probes based on the 5´ flap endonuclease activity.

HOT FIREPol® DNA Polymerase is activated by a 12 min incubation step at 95°C. This prevents the extension of non-specifically annealed primers and primer-dimers formed at low temperatures during qPCR setup.

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 12-15 min

Detection type: Probe-based

Reference dye: ROX

Compatible real-time instruments: Cyclers that require ROX reference dye. Check Your cycler!

Mix Components

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

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

dNTPsdATP, dCTP, dGTP and dTTP

ROX reference dye

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.

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: -20ºC

Temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of HOT FIREPol® Probe qPCR Mix Plus.

Shipping

At room temperature