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Fluorescein-C3-Suc-CPG Column (O-DMT-N-Fluorescein-3-aminopropan-1,2-diol-Suc-CPG)

Fluorescein-C3-Suc-CPG Column (O-DMT-N-Fluorescein-3-aminopropan-1,2-diol-Suc-CPG)

Fluorescein-C3-Suc-CPG Column (O-DMT-N-Fluorescein-3-aminopropan-1,2-diol-Suc-CPG), 500 Å, 1 µmol, Super

CPG column used to incorporate a 3'-fluorescein dye moiety with a C3 spacer into an oligonucleotide.

Key features

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  • Used to add a green-blue fluorescent dye to the 3' end of an oligonucleotide.
  • Maximal emission around 525 nm.
  • Quenched by BHQ-1, DDQ-1 and dabcyl.
  • Derived from the single isomer 5-carboxyfluorescein.
  • 500 Å CPG suitable for high yield applications such as therapeutic oligos (≤ 30mers).
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Item ID SCG5-5018-1
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.

Synthesizer
Column
Type/Description
Notes
MerMade 6,12
MerMade, syringe (all scales)
Pipette type column
A MerMade column is also known as a Supercolumn
MerMade 4, 48X, 96E, 192E, 192X
MerMade, Syringe (up to 1.3 mL)
Pipette type column
A MerMade column is also known as a Supercolumn
ABI 384 / 394
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
Expedite 8909
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
ABI3900
MerMade
Pipette type column
A MerMade column is also known as a Supercolumn
K&A H4, H8, H8SE, H2, H32, H64
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
K&A S4CL/S8CL
Luer
  Barrel column with luer fitting at either end 
 Also known as Standard. For this instrument, we recommend the Luer (Standard) column as the ALL-FIT columns have a wider barrel.
Dr Oligo 48
MerMade
Pipette type column
A MerMade column is also known as a Supercolumn
 Dr Oligo 192XLc, 768XLc just plates 
 MerMade, Syringe (up to 1.3 mL) 
Pipette type column
A MerMade column is also known as a Supercolumn
 OligoMaker X12, 48, 192, X192, X96 
MerMade, Syringe (up to 1.3 mL)
Pipette type column
A MerMade column is also known as a Supercolumn

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