PCR Master Mix Calculator
Scale a 25 µL PCR recipe across the plate with a pipetting overage — and keep the template out of the master mix, where the contamination fence wants it.
PCR Master Mix Calculator
Results recalculate instantly on every keystroke. Nothing you type is transmitted.
What this result does not account for
- Scales the standard 25 µL Taq recipe only
- No hot-start, GC-buffer or multiplex variants
In short: Ten reactions at a 10% overage: the master mix is 24 µL per tube scaled by 11 tube-equivalents — 264.000000 µL total — built of 10× buffer 27.500000 µL, 10 mM dNTPs 5.500000 µL, each primer 5.500000 µL, polymerase 1.375000 µL and water 218.625000 µL. At the 5 U/µL stock that enzyme is 6.875000 units across the run, 0.625000 per tube. The template — 1 µL per tube — never joins the mix: it is the per-tube seed behind the contamination fence.
Formula
mix = (25 − template) × N × (1 + overage) · each component × N × (1 + overage)
The NEB-style 25 µL recipe — 10× buffer 2.5, 10 mM dNTPs 0.5, 10 µM primers 0.5 + 0.5, polymerase 0.125 µL, template the variable, water to volume — multiplied by the tube count and the overage. The template is deliberately excluded from the multiplication: it joins each tube individually, one opening of the stock per tube, so a contaminated tip cannot inoculate the whole master mix.
Worked Example
- Enter the tube count and the overage percent.
- Enter template per tube and the enzyme stock.
- Read the master mix volume and the component bill.
- Add the template per tube, after the mix is aliquoted.
Defaults: 10 reactions, 10% overage → mix 264.000000 µL, water 218.625000 µL, enzyme 6.875000 U. A full plate (96, 10%) → 2,534.400000 µL of mix and 66.000000 U of enzyme. At 0% overage the ten-tube mix is 240.000000 µL — and one clingy pipette tip eats the margin.
Strengths & Limits Of This Model
Where this engine is strong
- Template kept outside the mix by design
- Enzyme units priced, not just microlitres
Where it stops
- One recipe, no chemistry variants
- No primer-stock concentration entry
Practical Use Cases
Plate runs
96-well shopping list
Teaching labs
recipe scaling with the fence
Protocol checks
units per tube audited
Methodology & Editorial Standards
Computation runs in IEEE-754 double precision at full internal precision; rounding to two decimal places occurs strictly at the display layer, so no cumulative drift enters the result. All monetary outputs use accounting presentation — grouped thousands, two decimals, negatives in parentheses — so figures can be transcribed directly into a model or working paper. Division-by-zero and out-of-domain inputs return an em-dash rather than a misleading number.
This engine was reconciled against an independent reference implementation and hand-verified for the worked example above before release. Our full five-stage review process is published on the About Us page.
Disclaimer. This calculator is provided for informational and modelling purposes only and does not constitute financial, tax, legal, medical, or engineering advice. Verify all figures with a qualified professional before acting on them.
PCR Master Mix Calculator — 8 Expert FAQs
8 analyst-written answers to the questions practitioners actually ask — optimised for voice and answer-engine retrieval.
Why does the template stay out of the master mix?
Contamination control. The master mix is one big open tube; the template is the one reagent that defines the amplicon, and a single airborne or tip-borne template molecule in the mix seeds every tube downstream. Adding template last, tube by tube, keeps a mistake local — one tube suspect, not the whole plate. The page prices the template separately for exactly that reason.
Why a 10% overage by default?
Pipettes under-deliver on viscous or tiny volumes, every tube keeps dead volume in its dead corners, and the last tube always comes up short without a margin. Ten percent is the bench convention that covers all three; the field takes any percent so a stingy 5% or a luxurious 15% is one click away. Zero percent is allowed — and the page lets the arithmetic warn you by simply showing how little slack that leaves.
Which recipe is the page scaling?
The standard 25 µL Taq recipe as published by NEB: 10× buffer 2.5 µL, 10 mM dNTPs 0.5 µL, 10 µM forward and reverse primers 0.5 µL each, Taq 0.125 µL, template the variable, water to 25. The 50 µL column of the same protocol is exactly double — scale this page’s output by two and the bill holds. High-GC or long amplicons change the recipe, not the multiplication.
How many units of polymerase is that per tube?
0.625000 U per 25 µL at the standard 5 U/µL stock — the same ratio NEB prices as 1.25 units per 50 µL. The enzyme ledger prints both the stock-dependent total and the per-tube figure, because the units are what the vendor catalog actually sells: a 500-unit vial stocks 800 of these ten-tube runs.
Can I change the reaction volume?
The page scales the 25 µL standard; any other volume is a proportional recipe swap — a 50 µL reaction doubles every per-tube volume including the template. What must NOT scale linearly is the enzyme if you change chemistry: different polymerases have their own unit recommendations, and the stock field is there so the ledger stays honest when the vial in your hand is 1 U/µL instead of 5.
Why does the page refuse huge reaction counts?
Past a few thousand tubes the arithmetic is still right but the workflow is wrong — nobody pipettes 5,000 reactions from a master mix in tubes; that is a production line with plate robots and vendor kits. The refusal names the boundary honestly instead of printing a 12-digit microlitre number that no bench could use.
What is the contamination fence, exactly?
The discipline of one-way flow: pre-PCR mix assembly in a clean area, template added in a separate one, amplified product never brought back. The mix calculator supports it structurally — the template is priced per tube, last — because the arithmetic of a master mix and the hygiene of one are the same lesson: minimize the number of times the template stock is opened.
Does the overage multiply the template too?
No — deliberately. The overage exists because the mix is distributed from one tube into many, losing a little at every transfer; the template is pipetted straight into each tube with no intermediate distribution, so it needs no margin. Pricing template × N without overage is the honest bill for how the bench actually works.