Freedom fluorescein dyes
6-FAM (Fluorescein): A single isomer derivative of fluorescein. FAM is a commonly used fluorescent dye attachment for oligonucleotides and is compatible with a variety of 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.
SUN: A VIC® (ThermoFisher Scientific) equivalent (emission maximum of 554 nm) that is recommended for use in multiplexed assays when added to the 5' end of a dual-labeled 5' nuclease probe for qPCR.
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.