BioDeviceHub

PCR Master Mix Calculator

Calculate the volume of each PCR component per reaction and for the whole master mix, including extra for pipetting loss, from your stock and final concentrations.

Formula

Vrxn,i=Vrxn×Cfinal,iCstock,iV_{\text{rxn},i} = V_{\text{rxn}} \times \dfrac{C_{\text{final},i}}{C_{\text{stock},i}}
Vwater=Vrxn−Vtemplate−∑VotherV_{\text{water}} = V_{\text{rxn}} - V_{\text{template}} - \sum V_{\text{other}}
Vmix,i=Vrxn,i×Nrxn×(1+extra100)V_{\text{mix},i} = V_{\text{rxn},i} \times N_{\text{rxn}} \times \left(1 + \dfrac{\text{extra}}{100}\right)
extra\text{extra}
percentage added to cover pipetting loss, commonly 10%

How it works

A master mix contains everything common to a set of reactions, so each tube receives an identical mixture and only the template differs. Each component volume follows from C₁V₁ = C₂V₂, and water fills the remainder.

Template is left out of the master mix and added to each tube. The extra percentage scales every component so that the last tube is not short.

Worked example

Eight 25 µL reactions with 10% extra and 1 µL of template each, using 10× buffer, 10 mM dNTPs, 10 µM primers at 0.4 µM final, and 1.25 U of a 5 U/µL polymerase.

  1. Per reaction: buffer 2.5 µL, dNTPs 0.5 µL, each primer 1 µL, polymerase 0.25 µL.
  2. Water = 25 − 1 − 5.25 = 18.75 µL.
  3. Scale by 8 × 1.1 = 8.8 reactions.

Make 211.2 µL of master mix and dispense 24 µL per tube, then add 1 µL of template to each.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Stock concentrations are as labelled.
  • The buffer either contains magnesium or you enter the amount to add.
  • Every reaction uses the same primers. Primer-specific mixes need the primers left out of the shared mix.

Common mistakes

  • Adding the polymerase first or leaving the mix warm. Assemble on ice and add the enzyme last.
  • Making exactly enough for the number of tubes. Pipetting loss will leave the last tube short.
  • Forgetting a no-template control when counting reactions.

Related tools

Primer Tm Calculator (Four Methods)Primer melting temperature by four accepted methods, side by sideOpen calculatorDNA Dilution CalculatorDilute a DNA or RNA stock to a working concentrationOpen calculatorqPCR ΔΔCt CalculatorRelative gene expression by the 2^−ΔΔCt methodOpen calculatorDilution Calculator (C1V1 = C2V2)Stock and diluent volumes for any dilutionOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.