The script above uses Beer's Law and the extinction coefficient of the Cy dyes to calculate the amount of Cy dye in a sample. The concentration is then used to calculate the Mass. The ratio of the mass of Cye-labelled nucleotides incorporated to the mass of cDNA synthesized is called the incorporation efficiency.

Beer's Law

A = E c l

A = absorbance E = extinction coefficient, units are /(M*cm) c = concentration l = pathlength

Extinction Coefficients Cy3 150000/M*cm Cy5 250000/M*cm

Mass of incorporated Cy nucleotide

mass (ug) = cuvette volume (uL) * concentration of Cy nucleotide (M) * FW of Cy nucleotide (g/mol)

Formula Weight
Cy3	1152 g/mol
Cy5	1178 g/mol

Mass of cDNA

ug cDNA = (OD260) * (33 ug/ml*OD) * (cuvette volume)

This calculation is only a rough measure by which to compare probes. It can be used whether one is labelling cDNA by direct incorporation of Cy-dNTP, or by conjugation of Cy dyes to aa-dUTP. In addition most spectrophotometers are linear only within a particular OD range, so the calculation can suffer from OD readings for the Cy dyes vs the cDNA that fall outside this range (for instance an OD reading of 3, if diluted 10-fold would read higher than 0.3 because many specs are linear only within a range of 0.1 - 0.8, but don't sweat it, it's not that important).