How to calculate a PCR master mix

Updated October 30, 2026

A master mix combines everything shared between your PCR reactions in one tube, so each reaction gets identical amounts and you pipette far fewer small volumes. The calculation has two parts: volumes per reaction, then scaling up.

Step 1: Volume of each component per reaction

For every component with a stock and final concentration:

volume per reaction = final concentration ÷ stock concentration × reaction volume

A typical 25 µL reaction:

Step 2: Scale up with extra volume

Liquid is lost on tip walls, so make mix for a few more reactions than you need. With 10% extra, 12 reactions become 13.2. Multiply every component except the template by 13.2:

• Buffer 33 µL, dNTPs 6.6 µL, each primer 16.5 µL, polymerase 3.3 µL, water 240.9 µL
• Master mix total: 316.8 µL. Dispense 24 µL into each tube, then add 1 µL template.

Good practice

• Add water first and polymerase last, and keep the mix on ice.
• Mix by gentle pipetting or flicking, then spin down. Don't vortex enzyme-containing mixes hard.
• Always include a no-template control (water instead of template) to catch contamination.
• Check whether your buffer already contains MgCl₂ (often 1.5 mM). If you add extra Mg²⁺, include it as its own line.