PCR Master Mixes and Supermixes
A PCR master mix is a premixed concentrated solution that has all of the components for a real-time PCR reaction that are not sample-specific. A master mix usually contains a thermostable DNA polymerase, dNTPs, MgCl2, and proprietary additives in a buffer optimized for PCR. Only template, primers, probes (if being used), and water, to make up the volume, need to be added. One or more dyes may be included for fluorescent detection of product formation in real-time quantitative PCR (qPCR).
Bio-Rad's supermixes for real-time PCR are highly engineered master mixes optimized for a wide variety of PCR, qPCR, and reverse transcription qPCR (RT-qPCR) applications, featuring proprietary buffer formulations and advanced enzymes such as the patented Sso7d fusion polymerase. Our supermixes yield superior speed, specificity, sensitivity, and PCR inhibitor tolerance even with the most challenging templates and target sequences.
Choosing a PCR Master Mix
Using a PCR master mix for real-time PCR experiments provides faster setup with less pipetting, reduction in contamination, less tube-to-tube variability, and more reproducible results.
Bio-Rad PCR master mixes are optimized for different applications, for example, gene expression quantification, genotyping, or high-resolution melt analysis. There is a range of considerations when choosing the best PCR master mix for your application including:
- Type of PCR — screening, cloning, or quantitative
- Size of the amplicon
- Template complexity and G-C content, secondary structure, etc.
- Speed, fidelity, and processivity of the DNA polymerase
- Inhibitor tolerance
- Antibody-mediated hot-start polymerase
- Tolerance to reaction parameters such as different primer Tm values
- Scalability
- Cost per reaction
PCR Master Mix Considerations
Generally, the main considerations for choosing a PCR master mix for real-time PCR are the application, sample type and target sequence. Furthermore, there are two main methods for measurement of PCR product formation: probe-based assays and double-stranded (ds) DNA-binding dyes such as SYBR® Green and EvaGreen.
The most common dsDNA-binding dye found in real-time PCR master mixes is SYBR® Green I dye. An alternative dye is EvaGreen. The main difference between the two dyes is their saturation limits. SYBR® Green is considered a non-saturating dye, whereas EvaGreen is a saturating dye, meaning it binds thoroughly and evenly throughout the PCR amplicon. This enables EvaGreen to be used for high-resolution melt applications. Probe-based assays add an additional oligo to the reaction that enables fluorescence detection. Probe assays are often used when more than one target is interrogated in a single reaction (multiplexing), for example, in SNP genotyping.
Some qPCR systems also use a passive reference dye to enable normalization for non-PCR fluorescence variation, mainly well-to-well differences in fluorescence intensities due to design of the optical system. The passive dye most commonly found in master mix is ROX.
There are several different DNA polymerases available in PCR master mixes for real-time PCR. The choice depends on the challenges imposed by the sample type, target sequence, speed required, and the length of the amplicon. Bio-Rad's iTaq™ supermixes contain a polymerase complexed with an antibody for hot-start applications to prevent mispriming. SsoAdvanced™ supermixes contain a patented engineered recombinant fusion polymerase featuring the Sso7d dsDNA-binding domain, which stabilizes the polymerase-template complex, yielding greater processivity and robust performance even in the presence of PCR inhibitors.
Bio-Rad offers PCR master mixes for real-time PCR, called supermixes, to cover a full range qPCR applications. At Bio-Rad.com, you will find supermixes with SYBR® Green or EvaGreen, hot-start and fusion DNA polymerases, and specially formulated buffers and additives for superior performance.