PCR master mix calculator | reactions, concentrations, overage

How to use (3 steps)

Choose an example or enter reaction volume and reaction count (or 96/384 well plate).
Set overage (e.g., 10%) and components (2× mix, primers, etc.).
Per-reaction volumes, totals with overage, and a pipetting protocol are shown.

This tool calculates volumes only. Optimize conditions separately.

Inputs

Example (preset)

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Mode

Reaction setup

Reaction count type Reactions (wells / tubes)

Reaction volume preset Reaction volume (µL)

Overage

Include template in master mix (optional)

Templates are often added separately to reduce contamination risk and sample-to-sample bias.

Components (quick mode)

Master mix name

Master mix (stock ×) Master mix (final ×)

Forward primer (stock µM) Forward primer (final µM)

Reverse primer (stock µM) Reverse primer (final µM)

Use probe (optional)

Template (µL / reaction)

Water is calculated as the remaining volume to reach the reaction volume.

Components (advanced mode)

Include exactly one water (fill to volume). Turn off “include template in master mix” to add template separately.

In master mix	Component	Mode	Parameters

Table scrolls horizontally.

Auto-calculate on input

Results

Reactions (wells)

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Prepared reactions (including overage)

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Reaction volume

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Dispense per well (master mix)

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Total master mix volume

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Component table

Component	Per reaction (µL)	Total (µL)	Notes

Table scrolls horizontally.

“Separate” indicates components typically added outside the master mix (e.g., template).

Protocol (copy-ready)

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Share URL / Export

Share URLs include inputs (including components) and can be long.

How it's calculated

From concentration to volume: Using C1V1=C2V2, V1 = (C_final / C_stock) × Vtotal.
Fold concentration (2×/5×): V = (final_x / stock_x) × Vtotal (usually final = 1×).
Overage: Percentage is rounded up (ceil), prepared = ceil(wells × (1 + pct/100)).
Water (fill): Water = Vtotal − ΣV(component). Negative means an error.
Totals: Components included in the master mix use prepared count; separate components use wells count (toggleable).

This tool calculates volumes only; it does not guarantee experimental success.

FAQ

What is a master mix?

A master mix is a common reagent mix prepared for multiple reactions. It reduces pipetting steps and variability.

How much overage should I add?

A typical guide is 5–10% or +1 reaction. Adjust based on pipetting and reaction count.

How do I enter a 2× (or 5×) master mix?

Enter it as a fold concentration. For a 2× mix to reach 1× final, the volume is about half of the reaction volume (computed automatically).

Should the template be included in the master mix?

Often the template is added separately to reduce contamination risk and sample-to-sample bias. This tool defaults to separate addition (toggleable).

Why is the water volume negative?

The component totals exceed the reaction volume. Increase reaction volume or reduce component volumes (especially template).

I got a very small volume (e.g., 0.1 µL).

Such volumes are hard to pipette. Consider intermediate dilution or adjusting the reaction volume.

Can I use this for qPCR?

Yes. Enter a 2× qPCR mix and primer concentrations to compute total volumes.

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