How much overage should I add?

Commonly 5–10%. For few reactions or viscous mixes use the higher end; the extra covers volume left in tips and on tube walls so you can fill every reaction.

Should template go in the master mix?

Usually no — each sample has different template, so it is added per well. Put only the shared reagents (buffer, dNTPs, primers if common, polymerase, water) in the mix.

PCR master mix calculator

Scale a per-reaction PCR recipe to any number of reactions, with a pipetting overage. Enter each component’s per-reaction volume to get the total to mix.

Number of reactions

Pipetting overage (%)

Component	Per reaction (µL)	Remove

How it works

Formula

For each component, total volume = per-reaction volume × reactions × (1 + overage ÷ 100). The overage (typically 5–10%) adds a few extra reactions’ worth so pipetting loss does not leave you short on the last tube.

Worked example

A 20 µL reaction over 20 samples with 10% overage runs as 22 effective reactions. A component at 10 µL/reaction needs 10 × 22 = 220 µL; a 1 µL/reaction primer needs 22 µL; the whole mix totals 20 × 22 = 440 µL.

When to use it

When setting up more than a couple of PCR or qPCR reactions: make one master mix of everything common to all wells, then aliquot. It improves consistency and saves pipetting versus per-tube setup.

Sensible defaults

Start from the example recipe, then edit each component’s per-reaction volume, add or remove rows, and set your reaction count and overage. Template is usually added per-well, not in the shared mix.

FAQ

How much overage should I add?: Commonly 5–10%. For few reactions or viscous mixes use the higher end; the extra covers volume left in tips and on tube walls so you can fill every reaction.
Should template go in the master mix?: Usually no — each sample has different template, so it is added per well. Put only the shared reagents (buffer, dNTPs, primers if common, polymerase, water) in the mix.