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 λmax (the maximum absorbance wavelength for the particular dye; see Table 1 for λmax 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 εdye, εbase, C.F.260, λmax, and A260 unit values required for the following calculations.
Calculate Base: Dye Ratio
- Determine the absorbance of your labeled sample at 260 nm (A260) and at λmax (Adye). Refer to Table 1 for λmax values.
- The Label IT® dye will also contribute to the absorbance at 260 nm, which can be corrected by calculating Abase. Use the Correction Factor (C.F.260) values from Table 1 to calculate Abase.
Abase = A260 - (Adye * 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 λmax.
C.F.260 = A260 free dye/Aλmax free dye
- Calculate the ratio of bases to dye molecules as follows:
base : dye = (Abase * εdye) / (Adye * εbase)
Calculate pmol of Dye per μg of Nucleic Acid
Table 1: Extinction Coefficients, C.F.260 and λmax values for Label IT® fluorophores
- Determine the A260 and Adye values as described above.
- Calculate dye concentration as follows:
Dye concentration (mol/l) = Adye / εdye
- 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.
- Calculate pmol of dye per μg of nucleic acid as follows:
pmol dye in sample / μg nucleic acid in sample
||Extinction Coefficient of Nucleic Acid Bound Dye - εdye(M-1cm-1)
Table 2: Extinction Coefficients and A260 conversion units for nucleic acids
||Extinction Coefficient of Nucleic Acid - εbase (M-1cm-1)
||A260 unit (µg/ml)