Phosphoramidites containing derivatives of fluorescein are designed to take advantage of the multicolor detection capability of modern DNA sequencers and genetic analyzers. Dichloro-dimethoxy-fluorescein (JOE) exhibits unique spectroscopic characteristics when compared to other derivates, with absorbance and emission maxima at 525 nm and 548 nm, respectively. JOE-dT can be inserted into the desired sequence as a replacement for a dT residue. Internal fluorophores offer a convenient method for introducing dyes in a sequence without disrupting normal base pairing.
Coupling: 10 minute coupling time recommended.
Deprotection: Use Ammonium Hydroxide and deprotect as required by nucleobases. If AMA is used, a small amount of a non-fluorescent impurity will be formed. To eliminate this impurity, first deprotect with ammonium hydroxide for 30 minutes at room temperature, add an equal volume of 40% methylamine and then complete the deprotection as required by the nucleobases - e.g. 10 minutes at 65°C or 2 hours at room temperature for standard bases. Extended deprotection in ammonium hydroxide for 17 h at 55 °C also yields acceptable results.
Freezer storage, -10 to -30°C, dry
The table below show pack size data and, for solutions, dilution and approximate coupling based on normal priming procedures.