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What to Consider When Choosing a PCR Master Mix

What to Consider When Choosing a PCR Master Mix

A PCR master mix can provide efficiency, decreasing assay cost compared with a component-based approach

Carl Strayer, PhD

Carl Strayer, PhD, is a technical service scientist quality specialist at Promega Corporation. He holds a PhD in biology from the University of Virginia.

Q: What are some advantages or benefits of incorporating PCR master mixes into PCR assays?

A: A PCR master mix provides efficiency in PCR assay set up with fewer components to gather and prepare (thaw and mix), and fewer pipetting steps. Efficiencies can decrease assay cost compared to a component-based approach. Additionally, the use of a PCR master mix has important quality and QC benefits. Fewer steps in set up reduce the risk of error, and fewer assay components simplify lot-tracking and similar documentation requirements. 

Q: What factors should clinical labs consider when selecting a PCR master mix?

A: For clinical labs, choosing a vendor who provides cGMP manufacturing standards and one that has the experience and quality system in place to provide lot-to-lot consistency are key considerations. These requirements help to ensure that your assay performs well over the long term. 

Related to development of the master mix, a good question to ask is: Does the vendor allow you to test and optimize critical components in the master mix (e.g., Taq and Mg++ concentrations)? The lab should also consider whether it requires a master mix formulation to overcome inhibitors that may be expected in samples. The master mix should give robust performance that works at the extremes of the assay. A critical way to address these considerations is by performing so-called guardbanding experiments, which labs should plan on as part of their testing and validation.

Another consideration that might be overlooked is the final dispensed format of the master mix, which should minimize reagent waste. If the lab relies on automation, a formulation and format that is automation-compatible is also necessary.

Q: What challenges do labs sometimes face when it comes to their master mixes? What are the solutions?

A: A common scenario is the need to accommodate challenging samples in which inhibitors may be expected or direct sample input is required. In these cases, choosing a vendor with experience in formulation options that address these challenges is critical. We also find that labs may have previously adopted a master mix that gives good performance in initial trials, with standards at expected concentrations. But the performance may not have held up over time, in use with a variety of real-world samples, and with inevitable variations in other parameters (such as extraction and instrument). Making sure that appropriate guardbanding is performed during master mix development and validation is key. Promega’s PCR Optimization Kit and custom master mix process and support helps customers perform this critical step.

Q: What experience does Promega have developing and manufacturing PCR master mixes?

A: Promega has been developing and manufacturing PCR reagents for over 30 years, both through our catalog and as a custom and OEM provider. Our experience in reagent and assay development spans a wide variety of PCR assay types, including endpoint and  real-time PCR and massively multiplexed assays. We have extensive experience in reagent manufacture, including individual  components and master mixes. Promega has earned several ISO certifications and offers cGMP manufacturing for high-quality, consistent products. 

Q: What options does Promega have for customizing master mixes if a catalog option is not quite right?

A: Promega’s PCR Optimization Kit is a very efficient approach for discovering an optimal PCR master mix for a specific assay. This kit contains eight pre-formulated PCR buffers that span a range of reaction requirements. The kit also provides a separate tube of MgCl2 , and concentrated GoTaq® MDx Hot Start Polymerase, our cGMP Taq enzyme preparation. Customers follow a simple three-step protocol to survey the buffers in combination with various Mg++ and enzyme concentrations to find the best combination for your assay. We also offer lyo-compatible GoTaq® enzyme and buffers if the goal is a lyophilized reaction mix. Once the best formulation is determined, we provide a made-to-order 2X PCR Master Mix manufactured under cGMP.