5 must-dos when evaluating a PCR master mix reagent
By: Roche Life Science
Posted: August 08, 2015 | Lab Life - Real-Time PCR
When it comes to PCR, the success of your assay is dependent upon the quality and reliability of the reagents you use. Your experimental samples are precious and your work is important, therefore you should not have to spend unnecessary time and energy troubleshooting assay reagents or optimizing your master mix components. Furthermore, depending on the goals of your experiment, there are various master mix reagents available to streamline your protocol and reduce pipetting steps and variability.
Whether you are designing small- or large- scale PCR assays for cloning, genotyping, or gene expression quantification, the master mix you choose is an essential component of your experimental design. Here, we will describe the five must-dos when evaluating a PCR master mix:
1. Know your basic ingredients
A good PCR master mix should contain all of the components for your PCR reaction that are not specific to the sample itself. This includes a DNA polymerase (preferably heat-stable), magnesium chloride and dNTPs in a PCR optimized buffer (often with proprietary additives/enhancers). This means that only your template, primers, probes (if applicable), and water to volume should be added. Understanding these basic components is essential for choosing the best master PCR mix to fit your assay needs.
2. Master mix optimization
Not all master mixes are created equally, and understanding the differences in PCR master mix reagents can sometimes mean the difference between a successful assay or not. You should know whether the DNA polymerase in the master mix is hot-start compatible with the inclusion of anti-Taq antibodies or aptamer complexes, which can limit non-specific DNA amplification (or mispriming) in your reaction. There are also master mixes containing high fidelity polymerases that can increase yield and accuracy. Also, some PCR master mixes contain uracil-N-glycosylase (UNG) and dNTPs with dUTP to prevent contamination with carry-over amplicons by degrading PCR products from prior amplifications without degrading native nucleic acid templates.
3. Considerations for RT-qPCR
If you are performing real time quantitative PCR (RT-qPCR), your master mix could include a passive internal reference dye (such as ROX) for normalization of well-to-well fluctuations in background fluorescence. Additionally, if you are not using sequence specific DNA probes, your master mix will need to include a non-specific DNA binding dye (i.e. SYBRⓇ Green), which is a non-saturating dye ideal for single target identification, screening assays, or when running a limited number of samples or assays. Depending on the length of the amplicon, sample type and the speed and throughput level desired, there are various master mixes available to maximize performance and efficiency.
4. Test your master mix
If you're planning to embark on large-scale PCR reactions or higher throughput applications, then comparing your master mixes upfront for speed (both labor or automation as well as machine run time), efficacy, fidelity and cost per reaction, can save you significant time and money in the long run. While in the short term, it can be difficult to justify the expense and time for performing initial direct comparisons. But after thousands of reactions, knowing that you are utilizing the most efficient and cost-effective master mix for your applications will provide peace of mind and prevent unnecessary headaches and reagent changes later on.
5. Plan ahead
Purchasing your master mix in larger quantities can be much more cost effective. Therefore, it's important to be able to plan ahead and estimate how much reagents you will need. If you only need to run a few assays with sequence specific DNA probes, but you know you will be running large numbers or assays using non-specific DNA binding dye (i.e. SYBRⓇ Green), then you can order accordingly. Similarly, it is also important to know the shelf life and optimal storage conditions (2-8 °C or -20 °C) of your master mix in order to get the most from your reagents in the appropriate timeframe