ProbeMasterMix (2X) Low ROX for qPCR

Genaxxon bioscience

ProbeMasterMix (2X) Low ROX für die qPCR

Advantages at a glance

Contains 50nM ROX for qPCR with probes
Excellent efficiency and specificity
Effective amplification of GC-rich DNA sequences
Hotstart technology prevents primer dimer formation, requires no pipetting on ice, and no immediate processing
Enables simultaneous detection of up to four DNA targets in a single multiplex PCR reaction

€115.00*

Prices excl. VAT plus shipping costs

Volume

2 x 1.25 mL

10 x 1.25 mL

Product number: M3031.0100
Ready to ship today,
Delivery time 1-2 workdays

Shipment: on wet ice. Store at -20°C. For laboratory usage only!

Product information "ProbeMasterMix (2X) Low ROX for qPCR"

ProbeMasterMix low ROX optimised for realtime PCR assays in block systems that contains all components to perform quantitative PCR with the exception of primer and template DNA. This 2X master mix is ready-to-use and contains optimised amounts of all ingredients.

Multiplex PCR: Applications at Genaxxon and at customers site have shown that the qPCR Probe Mastermix can be used for the simultaneous detection of up to four DNA targets in the same PCR reaction. For further details please refer to the product manual or contact us: info@genaxxon.com.

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Advantages of the Genaxxon ProbeMasterMix:

Hotstart technology enables setup of PCR mixture at room temperatur
No pipetting on ice necessary anymore
No immediate further processing (PCR) necessary. The pipetted pre-PCR mixtures can be left at RT for up to 3 days.
Amplification of GC-rich templates
High yields
No Primer dimers

The Genaxxon SuperHot Taq DNA polymerase offers convenience and practicality, without compromising efficiency or specificity:

1. The chemical modification inhibits the polymerase, which prevents false priming at all temperatures tested, allowing for PCR reactions to be set up at RT (20°C to 25°C), and
2. "High mix stability allows pre-assemby of the plate in advance", which means PCR reactions can be prepared and stored at RT for up to three days before they are run, without affecting the results.

The Probe qPCR master mix without ROX contains all the necessary components in an optimized composition to carry out quantitative PCR.

chemically modified Taq DNA Polymerase
dATP, dCTP, dGTP, dTTP
50nM ROX
optimized reaction buffer
stabilizers and enhancers to enable even amplification of low copy number targets
the small aliquote size of 1.25mL simplifies handling and storage (less freeze-thaw cycles per aliquote)

Stability at +2°C to +8°C (refrigerator): minimum of 8 months.

This Mastermix is specially suited for the following instruments: Applied Biosystems® 7500, 7500 Fast and ViiA™ 7, QuantStudio™ instruments, Agilent Mx3000P™, Mx3005P™, Mx4000™ and AriaMx.

Further realtime PCR Mastermixes and realtime FAST PCR Mastermixes can be found here: qPCR (Classic qPCR > or FAST and Multiplex qPCR >).

FAQs

The Hot Start-modification of the polymerase inhibits its enzymatic activity at low temperatures and prevents non-specific amplifications in the first PCR cycles, which can drastically affect the qPCR results.

The Ct (Threshold Cycle) value in qPCR is a measure of gene expression and is determined based on the fluorescence curve. The 2nd derivative method is used to determine the maximum change in the second derivative of the fluorescence curve - i.e. the point at which the fluorescence change no longer increases - and thus the exponential amplification is terminated. The cycle in which this is the case is called the "quantification cycle (Cq)". Please note that the calculation of the Cq value can also be done by other methods. It is important that you always use the same method for your experiment.

ROX (5-Carboxy-Rhodamin-X) is a fluorescent dye that some qPCR machines use as a passive reference dye. This is done to account for fluorescence variations between wells, which can result from factors like material inconsistencies or imprecise pipetting.

In general, it's advisable to take advantage of this normalization option whenever possible. It's essential to determine whether your system requires a low (low ROX) or high (high ROX) concentration of ROX. However, if your cycler lacks a ROX option, there's no need to be concerned. A well-optimized qPCR typically doesn't require additional normalization through a passive dye.

Genaxxon provides qPCR master mixes with ROX concentrations that range from none to low or high, catering to all your needs.

Yes, the used plates and foils could influence the assay performance according to several scientific studies (e.g. Reiter and Pfaffl 2008: Effects of Plate Position, Plate Type and Sealing Systems on Real-Time PCR Results, Biotechnology & Biotechnological Equipment, 22:3, 824-828, DOI: 10.1080/13102818.2008.10817561).

In general, white qPCR plates (e.g. Genaxxon’s I2017) have a lower light scattering and thus a higher signal strength than transparent qPCR plates (e.g. Genaxxon’s I2004, I2010, I2031). If you have a weakly expressed target or a only small amounts of sample material, we recommend using a white qPCR plate.

Beside this, it is essential to use optically clear foils for your qPCR. We do not recommend using “normal” PCR foils as adhesive residues and transparency differences in the material may lead to inhomogeneity during your qPCR assay.

Genaxxon offers different qPCR foils: a strongly adhesive foil (I2012) that securely seal the plate immediately, and a pressure-sensitive foil (I2248) that adheres to the plate by applying strong pressure.

TaqMan probes are hydrolysis probes used in qPCR for measuring DNA levels. Hydrolysis probes are single-stranded DNA oligonucleotides labeled with a fluorescent marker at one end and a quencher at the other. The quencher is a molecule that suppresses the fluorescence of the marker when they are in close proximity. When the probe binds to the DNA target and the polymerase initiates amplification, its exonuclease activity cleaves both the fluorescent marker and the quencher. This action removes the quencher's inhibitory effect. Since this process repeats in every cycle, the strength of the fluorescence signal, measured after excitation, is directly proportional to the amount of amplified DNA.

Hydrolysis probes (TaqMan) excel in applications such as SNP genotyping, splice variant analysis, and mutation detection using qPCR. In contrast, SYBRGreen-based assays are ideal for gene expression or miRNA expression analysis and offer additional advantages, including quality metrics like melting curve analysis.

Several scientific studies suggest that a properly optimized SYBRGreen assay is as effective as a hydrolysis probe (TaqMan) for gene expression analysis (e.g., Tajadini et. al. 2014: Comparison of SYBR Green and TaqMan® methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes, Advanced Biomedical Research, 3, 85, DOI: 10.4103/2277-9175.127998).

Key factors for success in both cases include selecting appropriate primers, optimizing the qPCR master mix, and ensuring high-quality, non-fragmented cDNA, which is crucial for TaqMan® probes as well.

Please not that the performance of a qPCR assay utilizing (TaqMan) probes is heavily influenced by the amplicon length, affecting its robustness, sensitivity, and specificity. In contrast, an assay with SYBRGreen demonstrates greater tolerance for variations in amplicon lengths.

To confirm specificity, you can perform post-qPCR analysis by either running the reaction mix on an agarose gel or, if you're using SYBRGreen, generating a melting curve. These methods will quickly indicate the presence of any unintended byproducts.

Just like any experiment, it's crucial to include the right controls in every qPCR assay. Depending on the assay format, we recommend the following controls:

No Template Control (NTC): For every gene being analyzed, it's important to incorporate an NTC (simply using water instead of a template). NTCs play a critical role in detecting cross-contamination and should be a standard part of every qPCR.
Reference Genes (formerly known as Housekeeping Genes): To ensure a robust gene expression analysis, it's a good practice to include multiple reference genes for each sample. These reference genes should exhibit stable expression regardless of any treatment, serving as reference points for subsequent relative quantification.
Alternative Positive Control: In cases where you're performing absolute quantifications and not using reference genes, including an alternative positive control is recommended. This control should consistently yield a known result in all scenarios.
noRT Control: If you're using reverse transcription (RT) from mRNA as the template, it's advisable to have a noRT control with a sample that lacks the enzyme.

Please note that a melting curve analysis is only possible when doing a qPCR with intercalating dyes, e.g. SYBRGreen, but not with probes.

The melting curve can be used to determine the specificity of a qPCR and to detect any by-products that may occur. It is therefore strongly recommended to perform a melting curve analysis for every qPCR when using an intercalating dye. To generate the melting curve, the qPCR cycler continuously increases the temperature and simultaneously measures the fluorescence. This drops as soon as the melting temperature of the amplicon produced during qPCR is exceeded.

Why? When the product melts, the intercalating dye is released, causing the fluorescence signal to drop. Usually, the melting curve is not shown directly as a fluorescence curve, but as a function of the change in fluorescence. A large change in fluorescence is represented as a peak (amplicons denature in this area).

Please note that a qPCR always shows only a single peak. Multiple peaks are - with the exception of multiplex assays! - a clear indication of unwanted by-products. These can, under certain circumstances, completely falsify the result and make it unusable.

Please note: When changing your master mix, the peak of a melting curve may shift. Why? The melting temperature also depends on the buffer used in the master mix, especially on the salt concentration. As different master mixes may use different buffer, it is quite likely that the resulting melting curve differs.

The concentration of primers plays a crucial role in qPCR performance and should be carefully optimized. Having a primer concentration that is too high or too low can negatively impact the calculation of the Cq value and may even encourage the formation of primer dimers (see e.g., Mikeska and Dobrovic 2009: Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays, BMC Research Notes, 2, 112, DOI: 10.1186/1756-0500-2-112). As a general guideline, you can initiate with a primer concentration of 100-200 nM. However, it's important to conduct test assays with both higher and lower concentrations to identify the optimal range.

The precise amount of template required for a specific qPCR is contingent on various factors. Excessive template can potentially inhibit the qPCR. In a typical qPCR, the range for template usage per reaction falls between 1 and 10 ng. Therefore, when initiating qPCR optimization, it's highly recommended to establish a template dilution series covering 5 to 6 orders of magnitude. This step is vital for determining the optimal template quantity. This dilution series assists in determining the primer efficiency, the potential for qPCR inhibition by the template, and the verification of whether the template quantity falls within the qPCR's linear range. Once these parameters are established, you can confidently select a template quantity that aligns with the linear range of the qPCR.

The specificity of a qPCR assay depends on several factors, including primer concentration, the melting temperature of the primers, annealing temperature and time, and the potential formation of primer dimers. If your qPCR assay lacks the desired specificity, we recommend the following:

Reduce the primer concentration.
Increase the annealing temperature (while ensuring it doesn't exceed the melting temperature of the primers).
Shorten the annealing time.

If none of these measures prove effective, consider redesigning the primers, exploring various options for length, melting temperature, and sequence.

SybrGreen has only a low specificity. It binds to double-stranded DNA during PCR cycles, even to potentially non-specific reaction products. This leads to inaccurate qPCR results with non-optimised protocols. To obtain more specific results, probe-based qPCR should be performed.

The master mixes from different manufacturers have different compositions, including the buffers. Some assays react sensitively to these variations, so that different results are possible. Therefore, a calibration is always necessary when changing the master mix. If you have further questions, please contact our technical support: info@genaxxon.com.

The protocols provided by Genaxxon are standard protocols. In order to obtain optimal specificity and amplification, individual optimisation of the experimental conditions is recommended. If you have further questions about this, please feel free to contact our technical support: info@genaxxon.com.

Depending on the qPCR instrument you’re using, you may need to use a passive reference dye, typically ROX, to overcome variations between wells caused by the machine limitations. Therefore, Genaxxon offers qPCR master mixes with either no or low and high amounts of the passive reference dye RO to ensure compatibility with a variety of qPCR instruments. If you have any problems choosing the right master mix for your application, please contact our technical support: info@genaxxon.com.

Genaxxon offers different qPCR master mixes optimised for fast qPCR assays. Depending on your needs, Genaxxon offers master mixes for fast qPCR assays with (GreenMasterMix FAST) or without (ProbeMasterMix FAST) a fluorescence dye and with no (No ROX) or a passive reference dye with different concentrations (Low ROX or High ROX). If you have any problems choosing the right master mix for your application, please contact our technical support: info@genaxxon.com.

Yes, you can switch to another qPCR master mix during your project or even in the middle of your experiment! You only need to perform a normalization of your qPCR beforehand. Please note that we generally recommend normalization for any qPCR. Also, check if you need to adjust the settings on your PCR cycler when changing the master mix.

Yes, all qPCR master mixes from Genaxxon can be used for multiplex applications. However, for easy and fast optimization of your multiplex reaction, we recommend using one of our specially designed multiplex master mixes, e.g., 5X qPCR Multiplex MasterMix (M3024), Multiplex HS MasterMix (2X) (M3013) or the lyophilized LyoPlex Multiplex PCR master mix (M3005).

2-time ready-to-use realtime PCR mastermix with 50nM ROX™ as reference dye.

Aliquot size: 1.25mL. This master mix is stable for at least 8 months at +2°C to +8°C.

Applikation / Application:

PCR Primer extension Multiplex PCR Low-copy targets PCR Real-time PCR Quantitative PCR

Einheiten / Units:

One unit is defined as the amount of enzyme which will convert 10 nmoles of dNTPs to an acid-insoluble form in 30 min at 72°C under the assay conditions (25 mM TAPS (tris-(hydroxymethyl)-methyl-amino-propanesulfonic acid, sodium salt) pH 9.3 (25°C); 50 mM KCl; 2 mM MgCl2; 1 mM b-mercaptoethanol; and activated calf thymus DNA as substrate.

Quelle / Source:

Sicherheits Hinweise / Safety

Klassifizierungen / Classification

eclass-Nr: 32-16-05-02

Documents - Protocols - Downloads :

Here you will find information and further literature. For further documents (certificates with additional lot numbers, safety data sheets in other languages, further product information) please contact Genaxxon biosience at: info@genaxxon.com or phone: +49 731 3608 123.

Documents:

Safety Data Sheet
Protocols
Certificate
Manuals
Spec. Product Description
General Data 2
General Data 3
Category List

Listed below are articles and references, in which the authors trust in the high quality of this Genaxxon product.
Source: NCBI PubMed

Fast PCR:

When performing fast real-time PCR, it is of high importance to consider primer design and target size. For efficient amplification during fast cycling conditions, target size between 70 bp and 200 bp is recommended. The shorter the target length, the faster the total run time. For other targets and other primer sets, the annealing step has to be determined experimentally.

Please use the following protocol for inspiration as you need to optimize instruments settings according to yourr target size and applied instruments.

Fast real-time PCR results using Genaxxon Master Mixes on Mx3005P
GreenMasterMix Low ROXTM and ProbeMasterMix Low ROXTM were tested on Mx3005P (Agilent Technologies) using fast cycling real-time 2-step protocols; Fast Pthr Green Mx and Fast Pthr Probe Mx, respectively.
qPCR Setup: For GreenMasterMix Low ROXTM and ProbeMasterMix Low ROXTM, primers targeting Pthr (75 bp) and 4 concentrations of gDNA (40 ng, 20 ng, 10 ng and 5 ng) were used. All DNA concentrations were tested in quadruple replicates. See fig. 3 and 4. The PCR reaction mix was run on MX3005P (Requiring Low ROX). Instrument settings, run times and quality criteria for the applied fast protocols; Fast Pthr Green Mx and Fast Pthr Probe Mx, were compared to the standard protocol; Standard Pthr Mx. See table 2.
Results: The standard Fast Pthr Probe Mx protocol has a run time of 83.4 minutes. Fast Pthr Green Mx and Fast Pthr Probe Mx protocols, resulted in run times of 50.1 and 46.8 minutes, respectively. All protocol times are inclusive ramping time and a15 minutes hot start. The determined criteria, as shown in table 2, for Fast Pthr Green Mx and Fast Pthr Probe Mx protocols were acceptable. Furthermore, the melt curve analysis using GreenMasterMix Low ROXTM with Fast Pthr Green Mx protocol, confirmed high specificity.

Figure 3. Amplification plot, standard curve and melt curve for GreenMasterMix run on Mx3005P using Fast Pthr Green Mx protocol. Primers targeting Pthr (75 bp) and 4 concentrations of gDNA (40 ng, 20 ng, 10 ng and 5 ng) were used. All DNA concentrations were tested in quadruple replicates.

Figure 4. Amplification plot and standard curve for ProbeMasterMix run on Mx3005P using Fast Pthr Probe Mx protocol. Primers targeting Pthr (75 bp) and 4 concentrations of gDNA (40 ng, 20 ng, 10 ng and 5 ng) were used.