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Fluorescein T Amidite

Fluorescein T Amidite

Phosphoramidite can be used for internally labelling an oligonucleotide with the fluorescein dye moiety.
  • Used to add a green-blue fluorescent dye internally or to the 5' end of an oligonucleotide.
  • Maximal emission around 525 nm.
  • Quenched by BHQ-1.
  • Has DMT functionality.
Item ID NACI1-017
TBD
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Product information

Fluorescein dyes fluoresce in the green-blue region of the visible spectrum, and are most efficiently quenched by BHQ-1. There are several ways of labelling an oligonucleotide with fluorescein-type dyes, and the choice of label is diverse, depending on the degree of chlorination of the aromatic rings. This determines the fluorescence emission of the dye. 5'-Fluorescein-CE Phosphoramidite (or “6-FAM”), derived from the single isomer 6 carboxyfluorescein is probably the most commonly used phosphoramidite, but 5'-Hexachloro-fluorescein-CE Phosphoramidite (HEX) and 5’ Tetrachlorofluorescein-CE Phosphoramidite (TET), can also be used to efficiently label an oligonucleotide at the 5'-end, with differing emission maxima. While both 6-Fluorescein-CE Phosphoramidite and Fluorescein-CE Phosphoramidite incorporate the same fluorescent dye as 6-FAM, the linking backbone differs. The former has a 1,3-diol structure, where the additional OH is protected with DMTr. This not only allows coupling efficiency monitoring by DMTr release, it allows the possibility of multiple additions within the oligo for use in, e.g. chromosome painting. However, this often requires a linker (e.g. spacer-18) to be incorporated between each addition to prevent self-quenching of fluorescein. In the same way spacer-C3 is used to mimic the distance between the 3’ and 5'-O of dR, the 1,3-diol arrangement of 6-Fluorescein-CE Phosphoramidite provides the same scenario. It must be noted that, as with spacer-C3, a distortion of the backbone occurs, particularly with multiple incorporations. Fluorescein-CE Phosphoramidite has a thiourea linkage which mimics the original method of incorporating fluorescein to an amino-modified oligo. It must be noted however that the linkage is attached via the 5 position of the ring system in this case. Internal sequence additions of Fluorescein are achieved using Fluorescein-dT. Again, multiple additions can be carried out but the spacing between each fluorescein-dT is crucial to prevent self-quenching. Labelling the 3'-end of an oligo with fluorescein can be achieved by a variety of solid supports, with spacer and dT options. Notably, the 3'-(6-FAM) CPG also allows the effective blockage of the 3'-terminus from polymerase extension, as well as exonuclease activity. Lastly, 5,6-Fluorescein-OH carboxylic acid product is a mixed isomer free acid which can be used for labelling free amines on proteins.

Properties:

  • Chemical Name: 5'-Dimethoxytrityloxy-5-[N-(6-carboxamido(3',6'-dipivaloylfluoresceinyl)-aminohexyl)-3-acrylimido]-2'-deoxyUridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl))]-phosphoramidite
  • Formula: C79H89N6O17P
  • Molecular Weight: 1425.26
  • Appearance: white solid
  • Absorption Maximum (Lambda Max): 498
  • Extinction Coefficient at Lambda max: 54700
  • Extinction Coefficient at 260 nm: 25500
  • Fluorescence Maximum: 520

Spectral properties measured in PCR buffer as internal labeled poly(T) oligonucleotide.

Product usage:

  • Coupling: 10-minute coupling
  • Deprotection conditions: With fast deprotecting amidites (ie. C-Ac, G-DMF, G-PAC) use concentrated NH4OH for 1 hour at 60 °C. With standard amidites (ie. C-Bz, G-iBu) use concentrated NH4OH for 5 hours at 60 °C. With base-sensitive fluorophores (eg. TAMRA) we recommend using t-butylamine/water (1:3) for 12-15 hours at 65 °C. The use of methylamine should be avoided with this product.
  • If the solution is not used during synthesis, it can be stored under argon and its functionality maintained up to 48 hours.Image of cleaved and deprotected structure:
  • The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 816.7

Storage and handling:

  • Shipping conditions: Cold
  • Storage conditions: -15 to -30 °C

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