In all of its grandeur, polymerase chain reaction (PCR) grants scientists power over genome. Generating ample supply of DNA segments from relatively small samples, PCR is an essential element of the biological sciences.
Whether you are a novice or a veteran, it’s important to address potential PCR pitfalls due to the finicky and often unpredictable nature of the reactions. Introducing a PCR master mix into the equation saves considerable energy and resources and contributes to the success of your PCR assay.
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What is a PCR Master Mix?
A PCR master mix is a ready-to-use premix containing the components to run a PCR assay. These components typically include a thermostable DNA Polymerase, dNTPs, MgCl2 and optimized reaction buffers for efficient PCR applications.
A master mix is often applied in high-yielding or routine PCR.
Why is a PCR Master Mix helpful?
Using a PCR master mix for PCR assays provides faster setup with less pipetting—the mix can be prepared once and divided among pipettes to save time.
By reducing the scope for pipetting, there is less room for experimental error, a reduction in contamination, and less variability among tubes.
A master mix is also advantageous because it provides consistency and more reproducible results. The concentrations are already optimized for efficient PCR amplification of DNA templates.
Furthermore, PCR commercial mixes are subject to quality control analysis for reliability and accuracy.
General Master Mix Recipe
A general master mix recipe for 50 µl reactions is displayed in Table 1. Note that this is a general recipe, and alterations may need to be made depending on specific uses.
If you begin with a premix solution, you would simply need to add your template DNA, primers, and nuclease-free water to a total volume of 50 µl.
How to Calculate the Total Volumes Needed for a Master Mix
The total volumes needed for a master mix varies by component and is calculated based on the total number of reactions you’re outputting. It is advised to follow the proper product literature from the supplier of your master mix.
To aid in the success of your large scale assays, GoldBio provides a master mix calculator that can be accessed:
https://www.goldbio.com/PCR%20Master%20Mix%20Calculator
To learn more about PCR Master Mixes, watch Goldbio's video below:
Tips Pertaining to Master Mixes
- Test master mix before applying to large quantities to test for speed, efficacy, and cost per reaction to ensure you’re utilizing the most effective master mix for your PCR assays.
- Use negative and positive controls when possible to identify any false positives or troubleshoot experimental errors.
- Add enhancers/stabilizers to improve amplification specificity and efficiency.
- Frozen PCR mixes may be advantageous for smaller-volume laboratories and unpredictable demand for PCR testing.
As PCR continues to alter the course of molecular and biochemical science, the fundamental framework remains the same. When one parameter changes, it may impact another.
Implementing the use of a master mix cuts down on tedious steps and reduces risk to optimize your PCR experiment.
References
Ginzinger, D. G. (2002, June 5). Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Retrieved April 16, 2020, from https://www.sciencedirect.com/science/article/pii/S0301472X02008068
Lorenz, T. C. (2012, May 22). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. Retrieved April 15, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846334/
Munson, E., Block, T., Voegeli, J. T., Hryciuk, J. E., & Schell, R. F. (2009, June 1). Cost-Effective Frozen Master Mix Modification of a Commercial Methicillin-Resistant Staphylococcus aureus PCR Assay. Retrieved April 17, 2020, from https://jcm.asm.org/content/47/6/1888.full
West, D. M., & Sawyer, J. (2006). Freezing Complete Polymerase Chain Reaction Master Mix Reagents for Routine Molecular Diagnostics. Journal of Veterinary Diagnostic Investigation, 18(6), 580–582. doi: 10.1177/104063870601800609
