Knowing the concentration of the DNA in a sample is fundamental in many experiments and laboratory techniques: with our DNA concentration calculator, you will learn how to determine that quantity using the absorbance.

Here you will learn:

  • What is DNA concentration, and how do we calculate it;
  • How to calculate the DNA concentration from A260 values;
  • The DNA concentration equation (both for nucleic acid samples and oligonucleotides).

Spectrophotometry for the calculation of the DNA concentration

The measurement of the concentration of DNA (or RNA) is obtained through various techniques: the choice depends on the desired sensitivity or purposes. Our DNA concentration calculator will analyze the spectrophotometric technique, commonly used since it doesn't require additional reagents and its relative simplicity, it lacks sensitivity at low concentrations, and it can't distinguish between the two nucleic acids.

The only instrument required for the measurement of the DNA concentration is a spectrophotometer. Place the sample in a quartz cuvette, and measure its absorbance at the ultraviolet wavelength of 260 nm260\ \text{nm}: we call this quantity A260\text{A}_{260}.

How to calculate the DNA concentration from the absorbance

The calculation of the DNA concentration from A260 values uses the Beer-Lambert Law. The equation for the DNA concentration is:

cDNA=A260bdfcc_{\text{DNA}} = \frac{\text{A}_{260}}{b} \cdot \text{df}\cdot c

Here we can identify:

  • cDNAc_{\text{DNA}}, the DNA concentration;
  • bb, the length of the optical path;
  • df\text{df}, the dilution factor; and
  • cc, the conversion factor.

The conversion factor depends on the type of nucleic acid under analysis. Its value is:

  • 33 µg/mL33\ \text{µg}/\text{mL} for single-stranded DNA (ssDNA);
  • 50 µg/mL50\ \text{µg}/\text{mL} for double-stranded DNA (dsDNA); and
  • 40 µg/mL40\ \text{µg}/\text{mL} for RNA.

The dilution factor is nothing but a measurement of the quantity of acid nucleic per unit volume of water.

How to calculate the concentration of any nucleotide sequence

The formula for the DNA concentration can be slightly modified to consider any short sequence of nucleotides (oligonucleotide), given that we know the composition of the sequence.

cDNAA260e260bwMdfc_{\text{DNA}} \frac{A_{260}}{e_{260}\cdot b} \cdot w_{\text{M}} \cdot \text{df}

Where we introduced the quantity:

  • e260e_{260}, the extinction coefficient; and
  • wMw_{\text{M}}, the molecular weight of the nucleotide strand.

To compute the molecular weight of the oligonucleotide, sum the weights of the nucleotides making up the sequence. First, use the data in the following table:

Nucleotide

ssDNA

dsDNA

RNA

Adenine

313.21 Da313.21\ \text{Da}

616 Da616\ \text{Da}

329 Da329\ \text{Da}

Guanine

329.21 Da329.21\ \text{Da}

617.88 Da617.88\ \text{Da}

345.21 Da345.21\ \text{Da}

Cytosine

289.18 Da289.18\ \text{Da}

617.88 Da617.88\ \text{Da}$$

305.18 Da305.18\ \text{Da}

Thymine

304.20 Da304.20\ \text{Da}

616.78 Da616.78\ \text{Da}

N/A

Uracil

N/A

N/A

306.20 Da306.20\ \text{Da}

The weights are expressed in Daltons, a unit defined by $$\text{N}{\text{A}}\ \text{Da}\simeq 1\ \text{g}/\text{mol}where where \text{N}{\text{A}}$$ is the Avogadro's number.

To refine the calculations for the weight of the oligonucleotide, you need to apply the following modifications:

  • Subtract 61.96 Da61.96\ \text{Da} for ssDNA or 123.38 Da123.38\ \text{Da} for dsDNA if you are using unmodified nucleotides.
  • Add 17.04 Da17.04\ \text{Da} for ssDNA or 34.08 Da34.08\ \text{Da} for dsDNA in nucleic acids with added phosphate groups.

*Add 159.0 Da159.0\ \text{Da} for RNA with added phosphate groups.

To calculate the absorption coefficient ϵ260\epsilon_{260} at 260 nm260\ \text{nm}, we use the nearest neighbour model for nucleotides. Use the formula:

And substitute the vales of absorption for the individual basis:

Adenine

Guanine

Cytosine

Thymine

Uracil

15,40015,400

11,50011,500

7,4007,400

8,7008,700

9,9009,900

And for the nearest neighbour in the 5'/3' direction, use the following table:

5'/3' position

Adenine

Guanine

Cytosine

Thymine

Uracil

Adenine

27,40027,400

25,00025,000

21,20021,200

22,80022,800

24,60024,600

Guanine

25,20025,200

21,60021,600

17,60017,600

20,00020,000

20,00020,000

Cytosine

21,20021,200

18,00018,000

14,60014,600

15,20015,200

17,20017,200

Thymine

23,40023,400

19,00019,000

16,20016,200

16,80016,800

N/A

Uracil

24,00024,000

21,20021,200

16,20016,200

N/A

19,60019,600

Use these values (expressed in M-1cm-1, the inverse of molarity per centimeter) in the DNA concentration formula to calculate the value of the DNA concentration from A260 values also in short sequences of nucleotides.

Davide Borchia
Sample type
single-stranded DNA
Conversion factor
μg/mL
Absorbance at λmax
Pathlength
in
Dilution factor
Concentration
µg/mL
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