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Oligo Resuspension and Dilution Calculator

Work out how much buffer to add to a dry oligonucleotide for a stock in µM, from its nmol or µg and molecular weight, and dilute that stock to a working concentration.

Formula

V (μL)=n (nmol)c (μM)V\,(\mu\mathrm{L}) = \dfrac{n\,(\mathrm{nmol})}{c\,(\mu\mathrm{M})}
n (nmol)=m (μg)MW (g/mol)×1000n\,(\mathrm{nmol}) = \dfrac{m\,(\mu\mathrm{g})}{\mathrm{MW}\,(\mathrm{g/mol})}\times 1000
V1=C2V2C1V_1 = \dfrac{C_2 V_2}{C_1}
nn
the yield on the oligo's data sheet, in nmol, or its mass and molecular weight
cc
the stock you want, usually 100 µM
C1, V1, C2, V2C_1,\ V_1,\ C_2,\ V_2
stock and working concentration and volume

How it works

Oligonucleotides arrive dry, with the yield printed on the tube. One nanomole dissolved in one microlitre is 1 mM, so dissolving N nanomoles in V microlitres gives N/V millimolar, which is 1000 N / V micromolar. Rearranged, the volume in microlitres for a stock in micromolar is 1000 times the nanomoles divided by the concentration, then divided by 1000 for the units: nmol divided by µM gives mL-equivalents of µL directly.

Most laboratories keep a 100 µM stock and dilute from it to the 5 to 20 µM working solutions used in PCR. The dilution is C₁V₁ = C₂V₂, and because concentrations are in the same unit the result is only a volume.

Worked example

A tube contains 25 nmol of a primer, and you want a 100 µM stock.

  1. Volume = 25 nmol / 100 µM = 0.25 nmol per µM = 250 µL.

Add 250 µL of buffer. To make 200 µL of a 10 µM working solution from that stock: 10 × 200 / 100 = 20 µL of stock plus 180 µL of buffer.

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

Assumptions

  • The stated yield is correct. Yields on data sheets are estimated from absorbance and can differ from the true amount.
  • The oligo dissolves completely, and its volume adds negligibly to the buffer volume.
  • For the µg option, the molecular weight is that of the oligo as supplied.

Common mistakes

  • Confusing nmol with OD units. They are different; an OD unit is an absorbance quantity, not a number of moles.
  • Adding the buffer without spinning the tube first, so part of the pellet stays on the cap.
  • Storing working dilutions for long periods in water at low concentration, where oligos are lost to the tube walls and degraded by nucleases.

Related tools

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Related equipment

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