PCR Master Mix Calculator
Enter the per-reaction volumes of every master-mix component, the number of reactions, and the excess fraction you want to cover pipetting losses. The calculator returns the total volume to prepare of each component — and the final master-mix volume — with practical warnings when your template or primer amounts cross well-known bench thresholds.
Calculator
Enter your recipe and calculate to see the master mix
Total master mix (excl. template)
Bench note
Per-component volumes
| Component | µL / rxn | µL total |
|---|
Inventory check (read-only)
Based on the Inventory page in this browser. BiomeBench only looks up stock here — it never consumes, reserves, or edits inventory.
Vcomponent = Cfinal × Vrxn ÷ Cstock · Vpolymerase = U ÷ (U/µL) · Vwater = (Vrxn − Vtemplate) − ΣV
Each additive's volume comes from the dilution equation C₁V₁ = C₂V₂. Polymerase is specified as an amount per reaction (U), so its volume is simply the desired units divided by the stock concentration. Water makes up the balance of the reaction volume after template and additives. Every volume is then scaled by (reactions × overage factor) for the master mix.
About this calculation
What this is
A PCR master mix is a single preparation that contains every reagent common to all reactions (buffer, dNTPs, polymerase, water), aliquoted before adding the template-specific primers. Preparing a master mix improves reproducibility and saves pipetting time.
Why it is used
Running 24, 48, or 96 reactions one reagent at a time is slow and error-prone. A master mix makes every reaction tube identical except for its template, and the dead-volume excess (typically 5–10 %) keeps the last tube from running short.
Why this calculator exists
Per-reaction volumes are tiny (µL) and small slips compound across the plate. The calculator multiplies each component by N and an excess factor, and surfaces common bench warnings (template percentage, primer concentration) that are easy to miss in a busy prep.
Key assumptions
- Every reaction uses the same master mix (same polymerase, same primer pair, same final volume).
- The excess fraction is appropriate for the number of reactions (a fixed 10 % works for ≤50 reactions).
- Component stocks are at the concentrations the calculator expects — verify before scaling.
Limitations
- It does not check primer Tm matching or dimer formation — design those separately.
- It does not model gradient cycler temperature optimisation.
- It does not adjust for viscous reagents (e.g. high-concentration glycerol stocks) where pipetting accuracy drops.
What this calculates
- Component volumes
- Volume of each additive per reaction and for the whole master mix.
- Nuclease-free water (total)
- Water volume to bring the master mix to the reaction volume.
- Total master mix volume
- Combined volume of all additives and water, excluding template.
Frequently asked questions
What is a PCR master mix, and why prepare one?
A master mix is a single bulk preparation containing every reaction component except the template DNA — buffer, dNTPs, MgCl₂, polymerase, primers, and water. Aliquoting one mix instead of pipetting each reagent into every tube keeps the reaction composition identical across samples and cuts pipetting error and time. This calculator scales a per-reaction recipe to any number of tubes and returns µL of each component per reaction and in total.
How do I use the PCR master mix calculator?
Enter the reaction volume, the number of reactions, the overage factor, and the template volume per tube, then set the stock and final concentration for each component: buffer (×/×), dNTPs (mM/mM), MgCl₂ (mM/mM), polymerase (U/µL stock with U desired per reaction), and both primers (µM/µM). The result shows the volume of each additive per reaction and for the whole master mix, with nuclease-free water filling the remainder. Enter 0 as the final concentration of an optional component, such as extra MgCl₂ or a primer, to omit it from the mix.
What is the overage factor, and why should I prepare extra master mix?
Pipetting loses a little of every reagent, so labs typically prepare 5–10% extra mix to keep the last tube from running short. In this calculator the overage is a multiplier applied to the whole recipe: 1.1 prepares 10% over, and accepted values run from 1 to 10. Every component and the water balance are scaled by number of reactions × overage, so the totals in the result are the volumes to pipet into the bulk tube.
How does the calculator work out the volume of each component?
Each concentration-based additive uses the dilution equation V = final concentration × reaction volume ÷ stock concentration, the same C1V1 relationship used at the bench. Polymerase is the exception: you enter an amount in units per reaction against a stock in U/µL, so its volume is units ÷ stock concentration. Water is balanced by subtraction — reaction volume minus template minus all additives.
Why is the template DNA not included in the master mix?
Template is the one component that differs between tubes, so it is added to each tube separately after the master mix is dispensed. You still enter the per-tube template volume, because the calculator subtracts it when balancing water so that mix plus template equals your reaction volume. If the additives do not fit in the space left after template, the calculator reports an error and asks you to increase the reaction volume or reduce component concentrations.
For research and educational use only. Not for clinical or diagnostic decisions. Always verify calculations independently before use in critical applications.