JOE phosphoramidite, 6-isomer

JOE phosphoramidite for oligonucleotide synthesis, pure 6-isomer (6-JOE). Fluorescent dye JOE is a fluorescein derivative containing two chlorine atoms and two methoxy groups. Its absorption and emission maxima are at 503 nm and 525 nm, respectively. By its spectral characteristics JOE is found in between FAM and TAMRA/ROX; therefore, this fluorophor is commonly used for multiplex detection, including that during DNA sequencing. Coupling time of 6 minutes. Deprotection can be perfromed under standard conditions using ammonium hydroxide; deprotection time depends on oligonucleotide composition and nucleobase protecting groups (deprotection for 17 h at 55 °C removes all protecting groups from standard nucleobases). AMA (solution of 30% ammonium hydroxide/40% aqueous methylamine 1:1 v/v) can be used with ~5% of non-fluorescent side product forming. To avoid formation of the side product, start deprotection with ammonium hydroxide (30 min at room temperature), then add an equal volume of 40% aqueous methylamine and continue deprotection as required with AMA (e.g. 10 min at 65 °C).