PCR · Reaction setup
PCR master mix calculator.
Scale a per-reaction recipe to N reactions plus overage, with water filled to the reaction volume.
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How to scale a PCR master mix
A master mix is every common component multiplied by the number of reactions, so you pipette each reagent once and avoid tube-to-tube error. Because some liquid is always lost to the tip and tube wall, prepare for extra reactions: master mix volume of each component = per-reaction volume x reactions x (1 + overage / 100). Water makes up the rest: water per reaction = reaction volume - sum of components - template.
Worked example. 8 reactions, 10% overage, 25 uL, NEB Taq recipe, 1 uL template. Multiplier 8.8. Buffer 2.5 x 8.8 = 22 uL, dNTPs 4.4 uL, each primer 4.4 uL, enzyme 1.1 uL, water (25 - 4.125 - 1) x 8.8 = 174.9 uL. Dispense 24 uL into each tube and add 1 uL template.
The default volumes are the 25 uL column of NEB's Standard Taq Buffer protocol (final 1X buffer, 200 uM dNTPs, 0.2 uM each primer). Other enzymes use different buffers and concentrations, so use the volumes from your own enzyme's protocol. Check template amount with the A260 concentration calculator.
Frequently asked questions
How much overage should I add?
Ten percent is a common allowance, and some labs use more for many reactions or viscous mixes. The calculator lets you set any percentage.
Does the template go in the master mix?
Usually not. Each tube gets its own template, so the master mix aliquot is the reaction volume minus the template volume.
Can I use this for qPCR or other enzymes?
Yes, as long as you enter the per-reaction volumes from that kit protocol. The defaults are for a standard Taq reaction only.
What if the components exceed the reaction volume?
The calculator reports an error. Use more concentrated stocks or a larger reaction volume.
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Research use and scope
This is arithmetic for research-bench planning, not clinical, diagnostic or safety advice. Conversion factors are standard approximations; strain, instrument, reagent and protocol differences matter. Check results against your own protocol, kit manual and lab supervisor, and follow your institution's biosafety rules.