6 steps to compare the performance of two different reaction mixtures for real-time PCR

# Real-Time PCR Mix Evaluation Guide When assessing a new real-time PCR master mix, it's crucial to consider the right parameters. This guide offers a straightforward protocol and tips for quickly evaluating a real-time PCR master mix. --- ## Step-by-Step Protocol for Evaluating Real-Time PCR Master Mixes ### 1. Prepare a DNA Dilution Series Start by preparing a dilution series as shown below. Mix the solutions gently by flicking the tubes (do **not vortex**!) and then spin them down.  For genomic DNA, include tubes 1–4. For other types of DNA, include tubes 1–6. The initial DNA concentration is 100 ng/µl. Final concentrations in each tube are indicated above the tubes. --- ### 2. Prepare a Reaction Mix Mix your reaction components according to the table provided below. After adding all components, briefly vortex the mixture for 2 seconds and spin it down. #### General Reaction Mix | Component | Volume (µl) | |----------------------|-------------| | Master Mix | 18 | | Primer Solution | 2 | #### For Probe-Based Assays | Component | Volume (µl) | |----------------------|-------------| | Master Mix | 17 | | Probe | 1 | | Primer Solution | 2 | --- ### 3. Distribute the Reaction Mix Transfer 20 µl of the prepared reaction mix into the bottom of the blue wells, as shown in the figure below.  --- ### 4. Add DNA Samples Add 5 µl of DNA from the dilution series into the corresponding wells. Ensure the pipette tip remains submerged as little as possible to avoid contamination. DNA should enter the liquid, not the sides of the wells. The numbers in the figure correspond to the tube numbers in the dilution series from Step 1. Add PCR-grade water to the no-template control (NTC) wells instead of DNA. --- ### 5. Run the Plate Run the plate on your real-time PCR instrument using the following cycling protocol: | Stage | Temperature (°C) | Time (s) | |-----------------------|------------------|----------| | Initial Denaturation | 95 | 10 | | Denaturation | 95 | 10 | | Annealing/Extension | 60 | 30 | | Repeat Steps 2-3 | 40 times | | After the run, generate the melt curve according to the instrument's default settings. --- ### 6. Analyze the Results The results must meet the criteria listed below to approve the master mix. Use the table provided to assess the mix. #### Key Parameters for Evaluation - **PCR Efficiency**: Should be between 90% and 110%. This is critical for determining the performance of the master mix. - **R² Value**: Should be ≥ 0.98, indicating good correlation between replicates. - **Standard Deviation**: ≤ 0.2, ensuring precise pipetting. - **Melt Curve**: One distinct peak at the intended melt temperature. Peaks in NTC wells should not match the product's melt temperature. - **Cq Values**: Should be consistent across replicates, reflecting proper amplification. --- ## Tips for a Thorough Evaluation ### Standard Curve Analysis Focus on three key aspects: PCR efficiency, R² value, and replicate consistency. Efficiency between 90% and 110% is ideal, while R² values ≥ 0.98 indicate strong data fitting. Replicate standard deviations ≤ 0.2 suggest accurate pipetting. ### Melt Curve Insights Look for a single distinct peak at the expected melt temperature. Variations of a few degrees are acceptable due to buffer and pH differences. Avoid peaks in NTC wells, which could signal contamination. ### Cq Values Cq values should align with efficiency and not be overemphasized alone. Extremely high Cq values may reflect low efficiency, which will surface in efficiency analysis. --- ## Additional Considerations When comparing two mixes, use identical primer concentrations and follow the same protocol. For probes, ensure no melt curve data is generated. Other factors to consider include primer/probe design, annealing temperatures, inhibitor presence, and fluorescent chemistry. Proper controls and threshold settings are equally vital. --- ## Extract from Ampliqon Products - **RealQ Plus 2x Master Mix Green Low ROX™ (A324499)** - **RealQ Plus 2x Master Mix for Probe Low ROX™ (A314499)** For further resources, explore our flyers or contact us directly for assistance. --- This guide provides a practical approach to evaluating real-time PCR master mixes. By focusing on key metrics and following the outlined steps, you can ensure reliable and reproducible results.

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