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Calculate Nucleic Acid Labeling Density

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

NOTE: If gel filtration (e.g. G50 columns included with some Label IT® kits) 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.

 

Measure the Absorbance at 260 nm and λmax

  1. Refer to Table 1 for λmax (the maximum absorbance wavelength) for your particular Label IT® fluorophore.
  2. Measure the absorbance of the labeled sample at both 260 nm and λmax. These values will be A260 and Adye, respectively.
  3. Estimate the number of fluorescent labels on the nucleic acid molecule using one of the methods below (Base:Dye Ratio or pmol of Dye/µg of Nucleic Acid).

 

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

FluorophoreExtinction Coefficient of Nucleic Acid Bound Dye
εdye (M-1cm-1)
C.F.260λmax (nm)
Cy®3150,0000.08550
Cy®5250,0000.05649
Fluorescein68,0000.32494
MFP48890,0000.1501
TM-Rhodamine100,0000.27546
CX-Rhodamine82,0000576

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

Nucleic AcidExtinction Coefficient of Nucleic Acid
εbase (M-1cm-1)
A260 unit (µg/ml)
dsDNA6,60050
ssDNA8,91933
DNA Oligo10,00033
RNA8,25040
ssRNA (21mer)9,70033

Calculate Base : Dye Ratio

  1. Determine the A260 and Adye values as described in Section ‘Measure the Absorbance at 260 nm and λmax.’
  2. 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)
  3. Refer to Table 1 and Table 2 for the Extinction Coefficients (ε) of the dye and nucleic acid, respectively. Calculate the ratio of bases to dye molecules using the following equation:
         Base : Dye = (Abase × εdye) / (Adye × εbase)

Calculate pmol of Dye / µg of Nucleic Acid

  1. Determine the A260 and Adye values as described in Section ‘Measure the Absorbance at 260 nm and λmax.’
  2. Calculate the concentration of dye (mol/L) in your sample with the following equation. Refer to Table 1 for εdye.
        Dye Concentration = Adye / εdye
  3. Convert to pmol of Dye by multiplying the Dye Concentration by the sample volume and appropriate unit conversion factors:
         pmol of Dye = Dye Concentration (mol/L) × Sample Volume (µl) × (1 L/106 µl) × (1012 pmol/mol)
  4. Refer to Table 2 for the ‘A260 conversion unit’ for your type of nucleic acid. Calculate the mass of nucleic acid (µg) in your sample using the following equation:
         Mass of Nucleic Acid = A260 × ‘A260 unit’ (μg/ml) × Sample Volume (ml)
  5. Finally, the pmol of Dye per µg of Nucleic Acid can be calculated by dividing as follows:
         pmol of Dye / Mass of Nucleic Acid 

Explore Related Info & Links

  • Tip from the Bench – Choosing a Label IT® Kit
  • Check out Label IT® Plasmid Delivery Controls in Fluorescein and Cy®3 ~ 1 label per 50-90 bp of plasmid DNA 
  • Check out Label IT® RNAi Delivery Controls in Fluorescein and Cy®3 ~ 1-2 labels per duplex

The TransMission
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