DNA Concentration Calculator

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.

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: we made a tool about it: the annealing temperature calculator) 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.

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:

where:

• $C$ – Concentration of the nucleic acid in the sample.

• $A_{260}$ – 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.

• $l$ – Pathlength, and more precisely, the length of the cuvette used. The standard value is 1 cm, but your instrument may use a different size.

• $\text{DF}$ – Dilution factor. It 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. The dilution factor calculator can help you determine the right value.

• $\text{CF}$ – Conversion factor, which 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 $A_{260}$, let's do the same for other sample types.

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:

where:

• $\varepsilon_{260}$ – Extinction coefficient; and
• $\text{MW}$ – Molecular weight.

The concentration units are like in the previous case. As you may have noticed, the conversion factor was replaced by $\frac{\text{molecular weight}}{\text{extinction coefficient}}$. 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 the steps below.

To find the total molecular weight of your oligo sequence, you simply need to 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 use for the calculation:

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.

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:

where:

• $\varepsilon_{260}$ – Extinction coefficient of the oligo sequence at 260 nm;

• $\textstyle\sum_{i=1}^{N-1}\varepsilon_{\text{nearest neighbor}}$ – Sum of the extinction coefficients of all adjacent pairs of nucleotides; and

• $\textstyle\sum_{i=2}^{N-1}\varepsilon_{\text{individual bases}}$ – 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:

Here 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:

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 $\varepsilon_{\text{nearest neighbor}}$ is:

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

This yields the extinction coefficient:

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.