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PCR Template Dilution Calculator

Work out how far to dilute a DNA template stock so that the volume you pipette carries the wanted amount, in ng or copies, with the stock and diluent volumes to prepare.

Formula

Cwork=mper rxnVtemplateC_{\text{work}} = \dfrac{m_{\text{per rxn}}}{V_{\text{template}}}
DF=CstockCwork\mathrm{DF} = \dfrac{C_{\text{stock}}}{C_{\text{work}}}
Vstock=VpreparedDFV_{\text{stock}} = \dfrac{V_{\text{prepared}}}{\mathrm{DF}}
mper rxnm_{\text{per rxn}}
ng or copies of template you want in each reaction
VtemplateV_{\text{template}}
volume of the diluted template added to each reaction

How it works

A reaction needs a certain amount of template, but the stock is usually far more concentrated than a pipette can measure directly. Diluting the stock to a working concentration lets you add a convenient volume, such as 1 or 2 µL, that carries the amount you want.

The working concentration is the amount you want divided by the volume you will add, and the dilution factor is the stock divided by that. Large dilutions, above about 1000-fold, are better made in two steps because the small stock volume becomes hard to pipette accurately.

Worked example

An 85 ng/µL genomic DNA stock; you want 10 ng in each reaction from a 2 µL template addition, and 100 µL of dilution.

  1. Working concentration = 10 ng / 2 µL = 5 ng/µL.
  2. Dilution factor = 85 / 5 = 17.
  3. Stock = 100 µL / 17 = 5.88 µL, plus 94.12 µL of diluent.

Make a 17-fold dilution: 5.88 µL of stock and 94.12 µL of diluent, enough for 50 reactions.

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

Assumptions

  • The stock concentration was measured accurately. Absorbance readings overestimate when contaminants are present.
  • Volumes add on mixing, which holds for dilute aqueous solutions.

Common mistakes

  • Forgetting that the stock's concentration is in ng per µL, not per mL, when entering a unit of µg/mL or g/L.
  • Diluting in water that can degrade low-concentration DNA. A buffered diluent is safer for storage.
  • Preparing exactly enough for the reactions with no allowance for pipetting loss.

Related tools

DNA Dilution CalculatorDilute a DNA or RNA stock to a working concentrationOpen calculatorPCR Master Mix CalculatorComponent volumes for any number of reactionsOpen calculatorDNA Copy Number CalculatorNumber of copies in a mass of plasmid, amplicon or genomeOpen calculatorPCR Annealing Temperature CalculatorOptimal annealing temperature from primer Tm and productOpen calculator

Related equipment

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