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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.
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.
Fluorophore | Extinction Coefficient of Nucleic Acid Bound Dye εdye (M-1cm-1) | C.F.260 | λmax (nm) |
---|---|---|---|
Cy®3 | 150,000 | 0.08 | 550 |
Cy®5 | 250,000 | 0.05 | 649 |
Fluorescein | 68,000 | 0.32 | 494 |
MFP488 | 90,000 | 0.1 | 501 |
TM-Rhodamine | 100,000 | 0.27 | 546 |
CX-Rhodamine | 82,000 | 0 | 576 |
Table 2. Extinction Coefficients and A260 conversion units for nucleic acids
Nucleic Acid | Extinction Coefficient of Nucleic Acid εbase (M-1cm-1) | A260 unit (µg/ml) |
---|---|---|
dsDNA | 6,600 | 50 |
ssDNA | 8,919 | 33 |
DNA Oligo | 10,000 | 33 |
RNA | 8,250 | 40 |
ssRNA (21mer) | 9,700 | 33 |
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