PCR Reaction Setup Calculator (with Multi-Sample Master Mix)

Setting up a PCR reaction is simply applying C₁V₁ = C₂V₂ to each component:

Volume of component = (final concentration / stock concentration) × total reaction volume
Water volume        = total reaction volume − sum of all component volumes

For multiple samples, prepare a master mix — do not add reagents tube by tube: Pipettes have a relative error exceeding 10% at small volumes (< 2 µL). Adding reagents tube by tube carries this error into every reaction; mixing once and then aliquoting introduces the error only once.

Prepare slightly more master mix than needed. The standard practice is to scale to n + 10% (this tool’s default), because tip dead volume and dispensing losses can leave the last tube short. With a small number of samples, prepare at least one extra tube’s worth.

Common mistakes:

• Mismatched concentration units. Primer stocks are typically 100 µM while the final concentration is 0.2 µM — a 500-fold difference; dNTP stocks are typically 10 mM while the final concentration is 0.2 mM. Unit mismatches are the most common source of errors.
• Do not add template to the master mix. Each tube has a different template; it must be added separately after aliquoting.
• Add the polymerase last; mix by gentle flicking, not vortexing. Polymerase is sensitive to shear forces.
• Commercial 2× Master Mix already contains buffer, dNTP, Mg²⁺, and polymerase. Only calculate volumes for primers, template, and water — do not add these components separately.