3 49 3 - A Guide to PCR Master Mix Preparation
November 20, 2021
3 49 2 - A Guide to PCR Master Mix Preparation

PCR master mix is also known as ready mix or super ix. It is a mixture of PCR components at the correct concentration that is prepared then divided into different PCR tubes. A master mix mostly includes buffer, MgCls, dNTPs. and DNA polymerase.

A master mix is used to reduces the possibility of contamination and reduce pipetting. A PCR master mix saves time and is convenient and prevents errors when mixing thus making it appropriate for different applications.

What is PCR Master Mix?

A PCR master mix is premix that is ready to use and contains elements to run a PCR essay. The elements include dNTPs, DNA Polymerase, MgCl2 and thermostable. A PCR master mix is also known as ready mix or super mix, a combination of PCR components at the best concentrations that can be prepared and distributed among the PCR tubes.

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A master mix allows the researcher to test various concentrations of RNA or DNA and set up controls without having to separately add exact amounts of cofactor, buffers, water and dNTP and cofactor to every reaction tube.

Therefore, master mix that contains all PCR components is prepared once. The correct amount of master mix is placed in plates or tubes. It is then combined with any elements that differ in the reactions including RNA or DNA primers or templates. A master mix is mostly applied to routine PCR or high-yielding.

Why is PCR Master Mix important?

PCR master mix provides less pipetting and quick setup for PCR assays. The mixture is prepared once and distributed in pipettes. This helps in saving time and there’s reduced chances of experimental errors or contamination. The mix is useful as it provides testable outcomes and consistency. PCR commercial mixes

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Types of Commercial Master Mixes

There are different PCR masters that are premade. The mixes help in avoiding errors in proportioning and measurements. Many commercial PCR mixes are selected for a specific PCR including hot start or well-suited for specific thermocyclers.

Commercial master mixes require preparation prior to using. They Mix will also need mixing through a centrifuge or pipette and also to be chilling, thawing and diluted with PCR-grade water. Ensure you select the right master mix that will satisfy your needs when doing experiments and PCR setup.

There are a variety of things to consider when selecting the PCR master mix that will suit your needs. This includes cost per reaction, types of PCR, inhibitor tolerance, scalability and much more.

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The main things to consider when selecting a PCR master mix is the sample type, application and target sequence. PCR product formation can be measured in two ways, double-stranded DNA binding dyes and assays.

Green I dye is the most common dsDNA binding dye that is found in PCR master mixes. The other dye is EvaGreen which can be used as an alternative dye. The salutation limits make the two types of dye different. Green is a non-saturating dye and EvaGreen is a saturating dye; this means that it binds consistently and the PCR amplicon. For this reason, EvaGreen is used for melt applications with high-resolution.

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Probe-based assays allow fluorescence detection through adding oligo to the reaction. Probe assays are commonly used when a target is incorporated in a single reaction for instance SNP genotyping. It also functions by recognizing a particular sequence on the preferred PCR product.

Preparation of PCR Master Mix

PCR helps in the production of millions of replicas of DNA sequence from few molecules. This approach requires much sensitivity to avoid the sample from getting contaminated with amplified products in the laboratory or other DNA.

Collecting the mixture, PCR process and DNA sample preparation should be conducted in different areas.  The reaction mixture can be prepared using a laminar flow cabinet. The experiment for reaction setup and DNA purification should be done with gloves.

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To perform the experiment prepares a master mix with buffer, water, primers, Taq DNA and dNTPs in one tube which is then distributed into separate tubes. The solution of template DNA and MgCl2 is then added.

This technique of setting reactions saves time through decreasing the amount of reagent transfers and also reduces the possibility of pipetting errors.

Components of PCR Master Mix

Template DNA

Template DNA is one of the essential ingredients for an effective PCR reaction. It is equally important as the dNTPs, polymerase, and DNA primers. Incorrect concentration of the template DNA in a PCR reaction can lead to reaction failure. Also a high concentration of DNA template in PCR can lead to an incorrect positive result.

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The Taq DNA polymerase is activated only through recognizing the template DNA for PCR reaction. The template DNA has to be bindes to the primer for polymerization to begin.

Primers

A primer is a single stranded DNA, short sequence commonly used in PCR experiments. Primers are also known as oligos or oligonucleotides. Primers are important elements for PCR amplification success. PCR specificity is determined by PCR primers.

dNTPs

There are four kinds of dNTP including guanine, cytosine, adenine and thymine. dNTPs is highly pure and used for all highly sensitive and standard PCR applications. These products are also useful in combination with all PCR enzymes such as HotStarTaq DNA Polymerase and Taq DNA Polymerase. dNTP mix can be used for various applications including Two-step RT-PCR, and genotyping,

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