What is a Real-time PCR Master Mix?

PCR master mix, as the name suggests, is a premixed solution of all reagents and essential components required to run a PCR assay. It contains dNTPs (dATP, dGTP, dTTP, and dCTP), Taq DNA polymerase enzymes, MgCl₂, stabilizers, and enhancers in a reaction buffer.

The PCR system has application in a broad range of molecular biology and biotech lab experiments, including cloning (or synthesis of specific DNA fragments), sequencing, genotyping, nucleic acid synthesis, gene expression, generation of NGS libraries, and mutagenesis.

A PCR master mix specifically helps researchers and scientists to enhance their PCR assay performance by providing a spectrum of benefits, including saving time and reducing the chances of any errors/cross-contamination in preparing PCR formulations. They are often utilized in routine or high-yielding PCR.

Commercial PCR master mixes are available in liquid and lyophilized forms. The liquid form mix is required to be stored at a temperature between -20°C – ­­4°C and is typically cheaper than the lyophilized or freeze-dried mixes.

Lyophilized PCR master mixes can be stored at ambient temperatures for a longer period. Moreover, they are easy to transport and while running PCR, the solution only needs to be reconstituted in the buffer solution, which comes with the master mix.

In this article, we will review the components of PCR master mix, its benefits in PCR applications, and the types of commercially available master mixes for different PCR reactions.

Why is a PCR Master Mix Beneficial?

The PCR master mix offers superior speed, sensitivity, specificity, and higher yield, even when the reaction is performed under stringent conditions, like with challenging target sequences or DNA templates. They provide more control over producing specific PCR products and prevent the reproducibility of undesired products.

Some other advantages of using PCR master mix are listed below:

• Despite measuring and adding all chemicals individually, the PCR master mix provides a premixed solution with all the necessary components at optimal concentrations for running PCR applications.
• PCR master mix saves preparation time for PCR amplification reactions.
• The master mix guarantees maximum consistency when preparing the PCR formulation, even while performing a high-volume assay.
• Using a PCR master mix reduces the pipetting steps, thus reducing the chances of contamination.
• As all the PCR components are already added in the PCR master mix, it reduces the chance of errors that are otherwise caused due to a missing component while preparing PCR formulations.
• The use of master mix reduces the variability among all tubes, which can occur while preparing the solution manually.
• Commercially available PCR master mix is subjected to quality control and analysis procedures, which assure the reproducibility of reliable and accurate PCR products.

PCR Master Mix Applications & Components

The PCR master mix helps researchers to streamline their PCR applications. In a master mix, the concentrations of DNA templates (either genomic DNA or cDNA) have already been optimized for efficient PCR amplification, thus accelerating the PCR system workflow.

The PCR 2X master mix is generally used in a volume of 25 µL, and the rest volume of 50 µL (differs depending on the experiment goal) is made by adding nuclease-free water, primers, and template DNA.

Given below are certain tips before using any PCR master mix in your real experiment:

• It is recommended to use positive and negative controls to troubleshoot experimental errors or detect false positives.
• Test a small quantity of the master mix for its efficiency, speed, and get an understanding of cost per reaction.
• You can add stabilizers or enhancers to enhance amplification efficiency and specificity.
• Frozen PCR mixes are a good option when laboratories cannot predict their needs or use PCR systems sparingly.

If you want to know if a specific master mix meets your needs and is quality assured, you have to check the production company’s website for certificates of analysis, brochures, guides, and Safety Data Sheets (SDS). For any questions, you can browse through its FAQs section.

Applications

Some common applications of PCR master mix include:

• High-throughput PCR
• Routine PCR
• DNA labeling
• Microarray analysis
• PCR-based DNA fingerprinting (RAPD, STR, and VNTR)
• High-specificity PCR
• Colony PCR
• Master mix having hot-start DNA polymerase, used for:

• Multiplex PCR
• Reducing primer dimers
• Setting up an ambient temperature that is compatible with automation

Components

A PCR master mix consists of a thermostable DNA polymerase, optimized reaction buffers, dNTPs, and MgCl₂ (or often MgSO₄). After getting a master mix, you are only required to add the following components before proceeding with your PCR experiments:

• Primers (forward and reverse)
• Buffers
• Template DNA
• Detection dye or fluorophore probes (only if needed)

Master Mix Types

A spectrum of PCR master mix is available suiting different PCR systems, including standard PCR, RT-PCR, and RT-qPCR.

The curated commercial master mix works best with the suitable size or type of DNA template. For example, some manufacturers offer standard PCR that works for amplification of DNA amplicons, ranging from 0.2-2 Kb. Some only work for GC-rich amplicons or amplify a 5-6 Kb amplicon.

Given below are some types of PCR master mix commercially available and their distinct features.

• High-Fidelity PCR: A combination of an Expand High-Fidelity PCR System and PCR-grade MgCl₂, dNTPs, and an optimized reaction buffer into a double-concentrated, ready-to-use product.

Some other types of high-fidelity master mix contain KOD Hot Start DNA polymerase with ultrapure deoxynucleotides, monoclonal antibodies, and reaction buffer with MgSO₄. High-fidelity polymerases have a very low error rate and highly increase the accuracy of the reaction.

• Routine type PCR master mix: Suitable to amplify a DNA fragment for approximately 5 Kb in size and having 40-60% GC content. It is further divided into two sub-categories based on the goal of running the PCR system:

• Hot start Taq: Can only function at normal cycling condition temperatures and is used to prevent undesired PCR product formation at a lower temperature.
• High-Fidelity Taq: Contains some specialized enzymes that can amplify amplicons of up to 20 Kb in size.
• Real-time qPCR master mix: For real-time PCR experiments, master mixes are designed to take advantage of dye and probe detection chemistry. Some master mixes already come along with added dye, such as SYBR® Green or any fluorophore-labeled probe. However, when it is not, you can add the desired dye in the master before use.

Often, some PCR instruments require a passive reference dye, such as ROX, to perform the real-time PCR. Therefore, it is necessary to understand your system requirements before purchasing the master mix.

Lab Equipment on Flexible Lease

PCR master mix consists of all reagents and chemicals required to run a PCR system. It has several advantages ranging from saving time and reducing errors to guaranteeing higher consistency in performing PCR reactions.

Master mixes are commercially available for use with different PCR systems, including qPCR, RT-PCR, real-time PCR, and standard and high-fidelity PCR.

To choose the perfect master mix for your experiment from a list of products available in the market, it is recommended to go through product details, specifications, application, and resources associated with the product. This also ensures higher reproducibility and efficiency of your PCR system.

Furthermore, to streamline your lab experiments, automated, cutting-edge instruments are often needed. However, purchasing requires a massive upfront investment many labs aren’t positioned to make.

In such a situation, Excedr can be your perfect lab partner. No matter the size or scope of your laboratory, Excedr’s customized leasing solution is affordable and fully customizable to fit any lab’s needs.

Contact us to learn more today.

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