How to Make a PCR Master Mix

Why use a master mix

Pipetting every component into every tube is slow and adds a small error each time. A master mix combines everything the reactions share in one tube, which is then split between the tubes before the template goes in. Each reaction gets the same mix, and you pipette far fewer small volumes.

Typical final concentrations

• Buffer: 1× (from a 10× or 5× stock).
• dNTPs: 0.2 mM each.
• MgCl₂: 1.5 to 2.5 mM, if the buffer does not already contain it.
• Primers: 0.1 to 0.5 µM each.
• Taq polymerase: about 1.25 units per 50 µL reaction.
• Template: 1 pg to 100 ng, depending on its source.

Follow your polymerase's manual where it differs; high-fidelity enzymes in particular have their own recommendations.

Calculating the volumes

For each component, volume per reaction = final concentration ÷ stock concentration × reaction volume. In a 25 µL reaction, 10× buffer needs 2.5 µL and 10 µM primers at 0.5 µM need 1.25 µL each. Water makes up the rest of the volume after the template.

Multiply every volume by the number of reactions plus about 10% extra, because some liquid is lost in tips and tube walls. For 10 reactions, make enough for 11.

Putting it together

1. Thaw the components on ice, then vortex and spin them briefly (but not the polymerase).
2. Add water first, then buffer, dNTPs, MgCl₂ and primers.
3. Add the polymerase last, mix gently by pipetting or flicking, and spin down.
4. Split the mix into the tubes, then add each template. Include a no-template control with water instead of template.

Calculators

The PCR Master Mix Calculator works out every volume for any number of reactions. Check your primers' annealing temperature with the Primer Tm Calculator, and make primer stocks with the Oligo Resuspension Calculator.