A PCR master mix is a premixed solution that contains most of the components necessary to run a PCR assay. The mix contains Taq DNA polymerase, dNTPs, MgCl2, as well as enhancers and stabilizers in a buffer that is optimized for DNA amplification by PCR.
Advantages of Using a PCR Master Mix
The major appeal for using a PCR master mix is its convenience. Most of the components have already been added and the formula has already been optimized, therefore significantly reducing pre-assay preparation time.
The use of master PCR mixes also ensures a high degree of consistency, even in high-volume assay environments, and the fewer pipetting steps involved also means fewer opportunities for contamination. Using a PCR master mix also reduces the chance for a preparation error, such as accidentally leaving out a component.
Additionally, commercial master mixes may contain various enhancers and stabilizers not familiar to novice PCR users. They are also subject to quality control procedures and analysis that produce an extremely accurate and reliable product. These factors result in overall enhanced assay performance.
All of these advantages are especially important to consider for users new to PCR.
What Do I Add to a PCR Master Mix?
- Detection Dye or Probes
- RNAse-free water
Master Mix Format & Stability
PCR master mixes commonly exist in two major formats. Most often, PCR master mixes come in liquid format. Liquid PCR master mixes generally require storage conditions between -20 °C – 4 °C, and tend to be less expensive than lyophilized mixes. They are thawed before use in a PCR assay.
PCR master mixes may also come in a lyophilized, or freeze-dried format. This allows the mix to be shipped at ambient temperatures. Some lyophilized master mixes may also be stored at ambient temperatures long-term. The master mix is then reconstituted with the accompanying buffer solution before use.
Routine PCR Master Mixes
Master mixes used for routine PCR assays typically amplify target sequences up to 5 kb in size, with a GC content ranging between 40% and 60%.
Various master mixes also exist for assays desiring an enhanced level of PCR performance:
- Hot Start Taq: prevention of non-specific amplification at lower temperatures
- High-Fidelity Taq: specialized enzymes for long-range amplicons, up to 20 kb
Real-Time PCR Master Mixes
Master mixes created for use with Real-time PCR assays are optimized for assays employing either dye or probe detection chemistry.
PCR master mixes used in assays employing dye detection chemistry may or may not contain the actual double-stranded DNA binding dye. If the dye is not included, the user may add an external custom dye to the master mix.
Similarly, PCR master mixes designed for assays utilizing probe detection chemistry do not include the fluorophore-labeled probe in the mix, but it may be added by the user.
PCR master mixes intended for assays using either type of detection chemistry may also include a passive reference dye; the most commonly included is ROX. This reference dye is necessary for the operation of certain, but not all real-time PCR instruments. Whether your instrument requires this reference dye should be determined prior to choosing a real-time PCR master mix.
Should I Ever Use a Home-Brewed PCR Master Mix?
In most cases, the advantages of using a PCR master mix are far greater than the benefits that may be gained from creating a personalized PCR master mix solution.
However, for assays requiring an enhanced level of user control over the experimental conditions, home-brewed PCR master mixes may be the superior choice. Downstream applications demanding this level of user control over the assay specifications may include sequencing, cloning, etc.
These home-brewed PCR master mixes often come at a reduced cost as well. Still, buffer optimization must be performed by the user, which involves an additional time investment.