If you're about to send your sample to a laboratory or happen to be simply interested in microbiology, the DNA concentration calculator might just come in handy. What's even better, it works for RNA and the oligo sequences as well!

We also strongly encourage you to read on if you wish to satisfy your thirst for knowledge. In this article, you will learn about DNA and RNA quantification, the average molecular weights of nucleotides, how to calculate DNA concentration from A260, and how to deal with oligonucleotides.

Spectrophotometric analysis of nucleic acids - DNA and RNA quantification

DNA and RNA quantification is usually performed prior to any experiments to determine the concentration and purity of the sample. Many reactions (such as PCR) involving nucleic acids have specific requirements regarding these two values, so determining them is necessary to obtain the expected results. The amount of added reagents also depends on the initial sample concentration.

The most popular methods of quantification are:

  • Spectrophotometric analysis of nucleic acids which works by measuring the UV absorbance of the substance to give you an idea of its concentration and if there are any contaminants. It doesn't require any additional reagents, but it can't distinguish between DNA and RNA and has limited sensitivity at low concentrations.
  • UV fluorescence tagging which uses dyes that fluoresce when they bind with nucleic acid. This method is more time-consuming and requires a set of known samples for comparison, but it's also more sensitive.
  • Agarose gel electrophoresis which is more complex, but it can also tell you if your sample is intact. It doesn't rely on checking the DNA absorbance. Instead, it uses agarose gel containing ethidium bromide and samples of known concentration for comparison. Then, a UV light is used to photograph the gel and you can compare fluorescence intensities and estimate the concentrations.

Omni's DNA concentration calculator is meant to help you analyze the results of the spectrophotometry.

How to calculate the DNA concentration from A260?

Luckily, if you're dealing with a standard sample, gathering data from the spectrophotometric analysis of nucleic acids is probably the most challenging part. After that, all you need to do is use the following formula derived from the Beer-Lambert Law:

concentration = (A260 / pathlength) * conversion factor * dilution factor,

where:

  • A260 - The maximum absorbance as indicated by the spectrophotometric reading. This usually occurs at the wavelength of 260 nm, but it may change depending on the nucleotide. So, if you wondered why is 260 nm used for DNA, this is the answer.

  • pathlength - Length of the cuvette used. The standard value is 1 cm, but your instrument may use a different size.

  • dilution factor - Applies only when the sample is diluted. For instance, if you diluted 1 liter of sample in 50 liters of H2O, the dilution factor would be 50.

  • conversion factor depends on the sample type:

    • 33 µg/mL for single-stranded DNA (ssDNA);
    • 50 µg/mL for double-stranded DNA (dsDNA); and
    • 40 µg/mL for RNA.

The most popular concentration units are μg/mL, ng/mL and mg/mL. After learning how to calculate DNA concentration from A260, let's do the same for other sample types.

How to compute the oligonucleotide sequence concentration?

Oligonucleotides are short, synthetic strands of DNA or RNA that have a number of applications in microbiology. Because they also can be used in processes such as PCR, it's sometimes useful to be able to calculate their concentration. This value is obtained from the equation:

concentration = (A260 / (extinction coefficient * pathlength)) * molecular weight * dilution factor.

The concentration units are like in the previous case. As you may have noticed, the conversion factor was replaced by molecular weight / extinction coefficient. Unfortunately, because oligos are short and can be made up of different nucleotides, estimates aren't accurate. Therefore, these have to be computed manually - we will take you through these steps below.

Average molecular weight of a nucleotide

To find the total molecular weight of your oligo sequence, you need to simply sum up the atomic weights of all nucleotides in it. You may have to adjust the value, depending on its type:

  • DNA without 5' monophosphate present - This is an oligo sequence without any modifications. To account for the removal of HPO2 and the addition of two hydrogens, subtract 61.96 Da for ssDNA or 123.38 Da for dsDNA.
  • DNA with 5' monophosphate present - To include 5' monophosphate left by restriction enzymes, add 17.04 Da for ssDNA or 34.08 Da for dsDNA.
  • RNA with a 5' triphosphate - To account for the 5' triphosphate, add 159.0 Da.

The unit used is Dalton, 1 Da ≈ 1 g/mol.

Below, we present the table of values you can to use for the calculation:

Nucleotide ssDNA dsDNA RNA
Adenine 313.21 Da 616.78 Da 329.21 Da
Guanine 329.21 Da 617.88 Da 345.21 Da
Cytosine 289.18 Da 617.88 Da 305.18 Da
Thymine 304.20 Da 616.78 Da N/A
Uracil N/A N/A 306.20 Da

So, for instance, if you tested an unmodified ssDNA oligo sequence AGGTC, its molecular weight would be:

313.21 + 2 * 329.21 + 304.2 + 289.18 - 61.96 = 1503.05 g/mol.

How to calculate the extinction coefficients of DNA and RNA oligo sequences?

The extinction coefficient of a material describes how strongly it absorbs light. This quantity is a bit tricky despite being a rather simple addition. This is because the sum depends not only on the component nucleotides but also on their order. So, how to calculate the extinction coefficient of your oligo sequence? Use the nearest neighbor model:

ε260 = Σ1N-1εnearest neighbor - Σ2N-1εindividual bases,

where:

  • ε260 - Extinction coefficient of the oligo sequence at 260 nm;
  • Σ1N-1εnearest neighbor - Sum of the extinction coefficients of all adjacent pairs of nucleotides; and
  • Σ2N-1εindividual bases - Sum of the extinction coefficients of individual nucleotides, excluding the first and the last ones.

The appropriate values can be obtained from the tables below. The units used are M-1 cm-1, where M stands for molarity.

Nearest neighbor values:

5'/3' position Adenine Guanine Cytosine Thymine Uracil
Adenine 27,400 25,000 21,200 22,800 24,600
Guanine 25,200 21,600 17,600 20,000 20,000
Cytosine 21,200 18,000 14,600 15,200 17,200
Thymine 23,400 19,000 16,200 16,800 N/A
Uracil 24,000 21,200 16,200 N/A 19,600

where 5'/3' position relates to the fact that each end of the DNA molecule has a number. The 5' carbon has a phosphate group attached to it, the 3' - carbon-hydroxyl group. DNA is read in from 5' to 3' direction.

Individual bases:

Adenine Guanine Cytosine Thymine Uracil
15,400 11,500 7,400 8,700 9,900

This may seem complicated, so let us continue the example of ssDNA oligo sequence AGGTC. It consists of 4 pairs: AG, GG, GT, and TC. Therefore, the sum of εnearest neighbor is:

Σ1N-1εnearest neighbor = 25,200 + 21,600 + 19,000 + 15,200 = 81,000 M-1 cm-1.

The individual bases to be considered are G, G, T. Hence

Σ2N-1εindividual bases = 2 * 11,500 + 8,700 = 31,700 M-1 cm-1.

This yields the extinction coefficient:

ε260 = 81,000 - 31,700 = 49,300 M-1 cm-1.

Since we have already calculated the molecular weight (1503.05 g/mol), if we assume the DNA absorbance to be 4,900, no dilution, and standard cuvette size, we can calculate the concentration. Inputting these values into the DNA concentration calculator gives the final result of 149.39 mg/mL.

FAQ

What is a good DNA concentration?

It can be anything between 10 and 300 ng/µL but there is no set value, and you should check with your laboratory. The DNA concentration required will depend on the following:

  • Sensitivity of the sequencing machine;
  • Sample size and volume; and
  • Sample type. For example, PCR products need less concentration than Plasmids.

What is OD₂₆₀?

OD₂₆₀ stands for the optical density at the wavelength of 260 nm, which measures the reduction in light transmittance caused by scattering. The slower the light is able to travel through a substance, the higher its OD₂₆₀ is. It's related to the absorbance, A₂₆₀, as follows:

OD₂₆₀ = A₂₆₀ * volume [ml] / pathlength [cm].

How to calculate the DNA concentration from OD₂₆₀?

You can calculate the DNA concentration using the formula:

concentration [μg/mL] = OD260 * conversion factor.

The conversion factor converts the optical density into concentration and has a fixed value for dsDNA, ssDNA, and RNA.

How to calculate the DNA yield from concentration?

To find the DNA yield from its concentration, use the following equation:

DNA yield [µg] = DNA concentration [μg/mL] * total sample volume [mL].

DNA yield also depends on the quality, freshness, and the type of the sample (e.g., saliva or blood).

What does the 260/280 ratio mean?

It is the ratio of the sample absorbance at the wavelengths of 260 and 280 nm. It is used as a measure of the purity of a nucleic acid sample. For pure DNA, the accepted value is ~1.8, whereas, for RNA, it's typically ~2.0.

How to calculate the 260/280 ratio?

To find the 260/280 ratio, proceed as follows:

  1. Measure the absorbance of the sample at the wavelength of 260 nm (A₂₆₀).
  2. Measure the absorbance of the sample at the wavelength of 280 nm (A₂₈₀).
  3. Divide A₂₆₀ by A₂₈₀ to obtain the ratio.

Why is 260 nm used for DNA?

Nucleic acids absorb UV light at a specific wavelength. For DNA and RNA, the maximum absorbance occurs at 260 nm. For comparison, at 280 nm, this value is approximately halved.

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