PCR Master Mix Calculator

How it works

volume of each component = reaction volume × final concentration ÷ stock concentration (dNTP: µM ÷ (mM × 1,000)) · polymerase (µL) = units ÷ (U/µL) · water = reaction volume − all components − template · master mix = (reaction volume − template) × reactions × (1 + extra %)

A master mix combines everything that is the same in every PCR tube — buffer, dNTPs, primers, extra magnesium, polymerase and water — so you pipette once and dispense equal volumes, which cuts pipetting error and contamination. Each volume follows C₁V₁ = C₂V₂. A typical Taq reaction uses 1X buffer (1.5 mM MgCl₂), 200 µM of each dNTP, 0.1–1 µM of each primer and 1.25 units of Taq per 50 µL (NEB protocol M0273). Making 10% extra covers the liquid lost on pipette tips; the template is added to each tube last.

Worked example

For 10 reactions of 25 µL with 10% extra (11 reactions prepared): 10X buffer 2.500 µL, 10 mM dNTPs 0.500 µL, each 10 µM primer 1.250 µL, 5 U/µL polymerase 0.125 µL (0.625 U), template 1 µL and water 18.375 µL per reaction. The master mix is 24 µL per tube, 24 × 11 = 264.0 µL in total.

Sources: NEB — PCR with Taq DNA Polymerase (M0273), protocols.io · Lorenz TC. Polymerase chain reaction: basic protocol plus troubleshooting. J Vis Exp 2012;(63):e3998

Frequently asked questions