Tips from the Bench: Nucleic Acid Labeling Tip

Calculate the Labeling Density of your Sample

Labeling density can be calculated easily using spectrophotometry for reactions with fluorescent Label IT® dyes. Depending on the sensitivity and accuracy of the the spectrophotometer used, this may require analysis of a large amount of the labeled sample (> 1 μg).

If gel filtration has been used to purify the labeled nucleic acid, make sure to perform an additional subsequent ethanol precipitation or silica membrane based purification to ensure accurate absorbance readings. While gel filtration is an effective purification procedure, erroneously high spectrophotometer readings (at 260 nm) have been observed when using this method.

To estimate the number of fluorescent labels on the nucleic acid molecule, measure the absorbance of the labeled sample at two different wavelengths - 260 nm and λ_max (the maximum absorbance wavelength for the particular dye; see Table 1 for λ_max of different Label IT® fluoropores). The labeling density can then be quantified using one of the following methods.

Note: Tables 1 and 2 below contain the ε_dye, ε_base, C.F.₂₆₀, λ_max, and A₂₆₀ unit values required for the following calculations.

Calculate Base: Dye Ratio

1. Determine the absorbance of your labeled sample at 260 nm (A₂₆₀) and at λ_max (A_dye). Refer to Table 1 for λ_max values.
2. The Label IT® dye will also contribute to the absorbance at 260 nm, which can be corrected by calculating A_base. Use the Correction Factor (C.F.₂₆₀) values from Table 1 to calculate A_base.
   A_base = A₂₆₀ - (A_dye * C.F.₂₆₀)
   Note: C.F.₂₆₀ is a constant value determined by dividing the absorbance of the free Label IT® dye at 260 nm with that at λ_max.
   C.F.₂₆₀ = A₂₆₀ free dye/A_λmax free dye
3. Calculate the ratio of bases to dye molecules as follows:
   base : dye = (A_base * ε_dye) / (A_dye * ε_base)

Calculate pmol of Dye per μg of Nucleic Acid

1. Determine the A₂₆₀ and A_dye values as described above.
2. Calculate dye concentration as follows:
   Dye concentration (mol/l) = A_dye / ε_dye
3. Calculate pmol of dye and μg of nucleic acid in sample:
   pmol dye in sample = dye conc. (mol/l) * 10¹² pmol/mol * sample volume (l)
   μg nucleic acid = A₂₆₀ * A₂₆₀ unit (μg/ml) * sample volume (ml)
   Note: A₂₆₀ unit is the concentration (µg/ml) of the nucleic acid solution that has an absorbance vallue of 1 at 260 nm. Refer to Table 2.
4. Calculate pmol of dye per μg of nucleic acid as follows:
   pmol dye in sample / μg nucleic acid in sample

Table 1: Extinction Coefficients, C.F.₂₆₀ and λ_max values for Label IT® fluorophores

Table 2: Extinction Coefficients and A₂₆₀ conversion units for nucleic acids

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