How can I increase the cDNA labeling efficiency when using the aminoallyl-method?
For most applications the optimal labeling level is about one dye molecule per 12 to 18. If the dye/base ratio is higher, hybridization efficiency may drop (due to steric hindrance by the dyes) and dye to dye fluorescence quenching may occur. The labeling efficiency is influenced by the reaction time/temperature, dye concentration, template purity, template concentration, type of dye etc. Free amines like Tris-buffer, ammonium acetate, hydroxylamine or proteins will interfere with the dye coupling and should be avoided. Make sure the pH (7,5-9) to guarantee optimal reactivity. Additionally, succinimidyl ester dyes hydrolyze spontaneously in the presence of moisture. It is important to keep the dyes anhydrous and dissolve it only in an anhydrous solvent (usually DMSO). Protect dyes from light (cover tubes with aluminum foil) and oxidation.
