About the PCR Master Mix Calculator
This PCR master mix calculator works out how much of each reagent to pipette when you set up many PCR reactions at once. Enter the reaction volume, the stock and final concentration of each component and the number of reactions, and it returns the per-reaction volume, the total volume for the whole master mix and how much water makes up the rest.
It is meant for molecular biology students and lab staff preparing standard Taq or high-fidelity PCRs, colony PCR screens or qPCR plates. Each component volume comes from the dilution equation C₁V₁ = C₂V₂, and the polymerase is dosed by units per reaction, which is how enzyme suppliers specify it. Template DNA is treated as something you add to each tube separately, so it is excluded from the master mix but counted in the reaction volume.
Pipetting viscous reagents always loses a little volume, so the calculator scales the mix by an overage (10% is typical) to make sure the last tube is not short. Defaults follow a common 50 µL Taq recipe: 1X buffer, 200 µM each dNTP, 0.5 µM of each primer and 1.25 U of polymerase. Always check the recommended concentrations in your polymerase manual — many buffers already contain 1.5–2 mM MgCl₂.
With the default inputs, the total master mix to prepare is 528 µL. Change any value above to recalculate instantly.
How to use the pcr master mix calculator
- 1Enter how many reactions you need, including positive and no-template controls.
- 2Set the reaction volume, the template volume per tube and an overage of about 10%.
- 3Check each stock and final concentration against your polymerase kit manual.
- 4Pipette the total volumes into one tube on ice, adding the polymerase last, and mix gently.
- 5Aliquot the per-tube volume into each PCR tube, then add template.
Formula and method
Each component volume per reaction comes from the dilution equation C₁V₁ = C₂V₂: the volume of stock needed equals the reaction volume times the final concentration divided by the stock concentration. A 10X buffer used at 1X in 50 µL needs 5 µL. The polymerase is dosed by activity, so its volume is the units per reaction divided by the stock activity in U/µL.
Water fills whatever is left after the reagents and the template. The master mix excludes the template, since each tube usually gets a different template. Every per-reaction volume is then multiplied by the number of reactions and by 1 + overage, so a 10% overage on 10 reactions makes enough mix for 11.
- V_rxn
- Final volume of one reaction (µL)
- C_stock, C_final
- Stock and final concentration of a component (same unit)
- U
- Polymerase units required per reaction
- N
- Number of reactions
- overage
- Extra fraction to cover pipetting losses
Worked examples
10 × 50 µL Taq reactions, 10% overage
Per 50 µL reaction you need 5 µL of 10X buffer, 1 µL of 10 mM dNTPs, 2.5 µL of each 10 µM primer and 0.25 µL of 5 U/µL Taq, leaving 36.75 µL of water after the 2 µL template. That is 48 µL of mix per tube; for 10 reactions plus 10% overage (×11) you prepare 528 µL.
24 × 25 µL reactions with added MgCl₂
A 25 µL reaction needs 2.5 µL buffer, 0.5 µL dNTPs, 1 µL of each primer at 0.4 µM, 1.5 µL of 25 mM MgCl₂ for an extra 1.5 mM and 0.125 µL polymerase, plus 17.375 µL water. Scaled by 24 × 1.1 = 26.4, the master mix totals 633.6 µL.
96-well qPCR-style setup, 20 µL, 5% overage
With a 2X ready-made mix that already contains dNTPs and enzyme, each 20 µL well needs 10 µL of 2X mix, 0.6 µL of each primer and 3.8 µL water, plus 5 µL template. For 96 wells with 5% overage (×100.8) you prepare 1,512 µL of master mix.
Frequently asked questions
How much overage should I add to a PCR master mix?+
Most labs add 5–10% extra, or simply calculate for one or two more reactions than they need. Enzyme and buffer stocks contain glycerol and stick to tips, so without overage the last tube often comes up short.
Should the template go into the master mix?+
Usually not. The master mix holds everything common to all reactions, and each tube receives its own template. If every reaction uses the same template with different primers, you can move the template into the mix and add primers separately instead.
What final concentrations are standard for PCR?+
A typical Taq reaction uses 1X buffer with 1.5–2 mM MgCl₂, 200 µM of each dNTP, 0.1–1 µM of each primer and 1–2.5 units of polymerase per 50 µL. High-fidelity enzymes often specify different values, so follow the supplier protocol.
When should I add extra MgCl₂?+
Only when your buffer is Mg-free or when optimising a difficult target. Most 10X Taq buffers already contain enough Mg²⁺ for 1.5 mM final. Too much magnesium lowers fidelity and promotes non-specific products; too little reduces yield.
Why add the polymerase last?+
Polymerase is the most sensitive and expensive component. Adding it last, keeping the mix on ice and mixing gently by pipetting rather than vortexing protects its activity and limits primer extension before cycling starts.