PCR Master Mix Calculator
Calculate volumes for each PCR master mix component scaled to your number of reactions, reaction volume, and overage factor.
Results
What is it?
A PCR master mix contains all reagents except the template DNA, pre-mixed for consistency across many reactions. This calculator scales a standard 25 uL reaction protocol (5 uL 5x buffer, 2 uL dNTPs, 1 uL each primer, 0.5 uL polymerase, 14.5 uL water, 1 uL template added separately) to any number of reactions and volume.
How to use
Enter the number of PCR reactions (always include at least one negative control), the reaction volume per tube, and the overage factor. The overage accounts for liquid lost during pipetting -- 1.10 (10%) is standard. Template DNA volumes are not included as they are added per tube.
Example scenario
Setting up 20 reactions at 25 uL with 1.1x overage: buffer = 22 x (25/25) x 5 = 110 uL, dNTPs = 44 uL, each primer = 22 uL, polymerase = 11 uL, water = 319 uL. Prepare the mix, aliquot 24 uL per tube, then add 1 uL template to each.
Pro tip
Always keep polymerase on ice and add it last to the master mix. For high-fidelity applications, use hotstart polymerases that activate only during the initial denaturation step, reducing non-specific amplification. Store unused master mix at -20C. Frozen aliquots are more reliable than repeated freeze-thaw cycles.