PCR Master Mix Calculator
Quickly calculate volumes for preparing PCR master mixes. Enter your reaction count and final volume, customize reagents, and scale for pipetting error. Ideal for batch PCR workflows.
Reaction Setup
Preparing for 27 total reactions
Reagents
Master Mix Volumes
| Reagent | Per rxn (µL) | Master mix (µL) |
|---|---|---|
| 10× PCR buffer | 2.00 | 54.00 |
| MgCl₂ | 1.20 | 32.40 |
| dNTPs | 0.40 | 10.80 |
| Forward primer | 0.80 | 21.60 |
| Reverse primer | 0.80 | 21.60 |
| Taq polymerase | 5.00 | 135.00 |
| Template DNA | 1.00 | 27.00 |
| Water | — | 237.60 |
| Total | 20 | 540.00 |
PCR Basics & Master Mix Strategy
A PCR (polymerase chain reaction) master mix is a pre-made cocktail of all reagents except template DNA. It simplifies workflows, reduces per-reaction setup time, and minimizes pipetting errors when running many samples in parallel.
Why Make a Master Mix?
- Speed: Mix once, dispense to all reaction tubes.
- Consistency: All reactions contain identical reagent ratios.
- Less pipetting: Reduces manual manipulation and cross-contamination risk.
- Higher success rate: Fewer opportunities for human error in concentration.
Common PCR Reagents
- PCR buffer (10×): Provides optimal pH and ionic strength; typically diluted 1×.
- MgCl₂: Essential cofactor for Taq; usual final concentration 1–2 mM.
- dNTPs: Building blocks for DNA synthesis; typical final concentration 0.2 mM (each).
- Primers: Define amplicon region; usual final concentration 0.1–1 µM.
- Taq polymerase: Heat-stable DNA polymerase; typical activity 1–2 units per reaction.
- Template DNA: Usually added per-tube to avoid cross-contamination.
Common Errors to Avoid
- Incorrect final concentration: Double-check stock concentrations and dilutions.
- Forgetting water: Water fills the remainder of the reaction volume.
- Thawing Taq on ice: Prevents premature extension; keep on ice until ready.
- Under-accounting for evaporation: Adding 10–20% extra accounts for dead volume and dispensing loss.
- Mixing master mix too vigorously: Can inactivate Taq; gently mix instead.
Master Mix in Microfluidics
Digital PCR and on-chip PCR rely on highly accurate partitioning of master mix into discrete droplets or chambers. Consistent reagent ratios are critical: a single off-ratio partitioning can invalidate an entire experiment. On-chip PCR typically uses 20–50 µL reactions in microfabricated channels, and master mix preparation is simplified because the device itself handles partition geometry.
Need a microfluidic device for PCR?
From rapid prototyping in 3D-printed resin to production-scale injection moulding in COC and COP. Optimize your digital PCR or on-chip PCR workflow.