Freedom fluorescein dyes
6-FAM (Fluorescein): A single isomer derivative of fluorescein. FAM is the most commonly used fluorescent dye attachment for oligonucleotides and is compatible with most fluorescence detection equipment. It becomes protonated and has
decreased fluorescence below pH 7; it is typically used in the pH range 7.5–8.5. FAM can be attached to 5' or 3' end of oligos.
6-FAM (NHS Ester): A post-deprotection reaction is used to attach this fluorophore to the oligonucleotide. This is commonly used to modify RNA oligos with FAM.
Fluorescein dT: Fluorescein is attached to position 5 of the thymine ring by a 6-carbon spacer arm.
HEX: Hexachlorofluorescein is a chemical relative of fluorescein that is commonly used for multiplexed assays with FAM and TET. HEX can be added only to the 5' end of an oligonucleotide.
JOE (NHS Ester): JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein) is available only as an NHS ester and must be attached to an amino-modified oligonucleotide. JOE is frequently used in multiplex reactions with FAM.
MAX (NHS Ester): Most often used to multiplex with FAM. It can be used as a VIC™ substitute because its spectral profile is nearly identical to VIC. Importantly, MAX is efficiently excited by either a 488 nm laser or a broad emission
light source; therefore, it can be used with most commercial detection platforms.
TET: Tetrachlorofluorescein is a chemical relative of fluorescein that is commonly used for multiplexed assays with FAM and HEX. TET can be added only to the 5' end of an oligonucleotide.