PCR Master Mix Calculator

About PCR Master Mix Calculator

This calculator scales a per-reaction PCR recipe up to the whole run. Enter the number of reactions, a percentage of extra volume and the total reaction volume, and it works out the amount of each reagent to combine, filling the rest with nuclease-free water. The reagent list is fully editable.

How to use it

1. Under Run setup, enter the number of reactions, the Overage percentage and the Rxn volume in microliters.
2. Edit the reagent names and per-reaction volumes in the Reagents list, using Add reagent or the trash icon to change the list.
3. Watch the multiplier badge, which equals reactions times one plus overage.
4. Read the Master mix table for per-reaction and total volumes, then use Copy table to paste it into a notebook.

Ten 25 uL reactions with 10 percent overage

Defaults: buffer 2.5, dNTPs 0.5, two primers 1 each, Taq 0.25, template 1 uL

Multiplier x11.00. Water 18.75 uL per reaction, 206.25 uL in the mix. Buffer 27.5 uL, Taq 2.75 uL.

When to use it

• Setting up a 96-well plate of identical reactions without pipetting each reagent 96 times.
• Adapting a polymerase manufacturer's recipe to your own reaction volume.
• Keeping a consistent, copyable record of the mix for a lab notebook.
• Adding an extra component such as DMSO or a probe to a standard mix.

Questions about PCR Master Mix Calculator

Why include overage at all?

Viscous reagents like polymerase stock cling to tips and tube walls, so a mix made for exactly N tubes runs short. Somewhere from 5 to 10 percent extra is common practice. The multiplier is N times one plus the overage fraction.

How is water worked out?

Reaction volume minus the sum of the listed reagent volumes. If those volumes add up to more than the reaction volume, water goes negative and a red warning appears.

Should template be in the mix?

The default list includes it, so it is scaled too. To add template to each tube separately, delete that row and lower Rxn volume to the mix volume per tube, which is 24 uL for a 25 uL reaction with 1 uL of template.

Does it handle reagent concentrations?

No. The concentration in a name such as dNTP mix (10 mM) is only a label. The tool works purely in microliters, so use the dilution calculator when you need to prepare a stock.

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