Oligo Synthesis : CEPs

Catalog Number: 10-5903-xx

Description: 5"-Tetrachloro-Fluorescein Phosphoramidite

FLUORESCEIN LABELLING

5’-Fluorescein phosphoramidite contains no 4,4’-dimethoxytrityl (DMT) groupand can be added only once at the 5’-terminus, thereby terminating synthesis.This product is prepared using the 6-carboxyfluorescein derivative. Thetetrachloro-, hexachloro-and dichloro-dimethoxy-fluorescein phosphoramidites aredesigned to take advantage of the multicolor detection capability of modern DNAsequencers and genetic analyzers. Fluorescein phosphoramidite is designed toproduce the same fluorescein-type structure as had been previously preparedusing fluorescein isothiocyanate (FITC). Our fluorescein phosphoramidite alsocontains a DMT group to allow quantification of coupling. The analogousstructure, 6-Fluorescein Phosphoramidite, prepared using 6-FAM, is alsoavailable, along with 6-Fluorescein Serinol Phosphoramidite. Fluorescein-dT canbe inserted into the desired sequence as a replacement for a dT residue.

We offer five fluorescein supports. Fluorescein CPG has traditionally beenused to add the fluorescein label at the 3’-terminus. The analogous structure,3’-(6-Fluorescein) CPG, prepared using 6-FAM, is now also available, along with6-Fluorescein Serinol CPG. We also offer 3’-(6-FAM) CPG and Fluorescein-dT CPG,both derivatives of 6-carboxyfluorescein (6-FAM). Both are single isomers anduse an amide linkage which is stable during cleavage and deprotection and doesnot allow isomer formation. 3’-(6-FAM) CPG allows effective blockage of the3’-terminus from polymerase extension as well as exonuclease digestion.Fluorescein-dT CPG allows both of these enzymatic activities to proceed. Normalcleavage and deprotection with ammonium hydroxide readily generates thefluorescein labelled oligos.

The spectral characteristics of these dyes are detailed on the right side ofthis page.

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MSDS

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Frequently Asked Technical Question

QUESTION: What are the relative extinction coefficients of 5"-Fluorescein, Hex and Tet etc.. at 260 nm and their Lambda max?

RESPONSE:Please see http://www.glenresearch.com/Technical/Extinctions.html

REFERENCE(S):Oligonucleotide Properties Calculator;http://www.basic.northwestern.edu/biotools/oligocalc.html

QUESTION: What are the relative extinction coefficients of various dyes?

RESPONSE:Please see http://www.glenres.com/Technical/Extinctions.html#dyes

QUESTION: Does AMA or methylamine cause any degradation to fluorescein or fluorescein-type dyes such as FAM or FITC?

RESPONSE:Response:While AMA (Ammonium hydroxide/40% Methylamine 1:1 v/v)is considered compatible with fluorescein, the use of methylamine when deprotecting a Fluorescein-labeled oligo does lead to a small amount of degradation, which is characterized by a the appearance of a late-eluting peak by RP HPLC that shows no visible fluorescein absorbance. With standard deprotection conditions (AMA 10 minutes at 65 C) the amount of degradation is approximately 5%.The impurity is not detected with AMA at RT for 2 hours.

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DILUTION/COUPLING DATA

The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures.

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