5'-Fluorescein Phosphoramidite

6-(3',6'-dipivaloylfluoresceinyl-6-carboxamido)-hexyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite

Product Specifications

Formula:
C46H58N3O10P
M.W.:
843.95
F.W.:
537.46
CAS Number:
204697-37-0
To Retrieve a Catalog Number, Select a Pack Size and Format:

Description

5'-Fluorescein phosphoramidite contains no 4,4'-dimethoxytrityl (DMT) group and can be added only once at the 5'-terminus, thereby terminating synthesis. This product is prepared using the 6-carboxyfluorescein derivative. The tetrachloro (TET)-, hexachloro (HEX) - and dichloro-dimethoxy (JOE)- fluorescein phosphoramidites are designed to take advantage of the multicolor detection capability of modern DNA sequencers and genetic analyzers. Fluorescein phosphoramidite is designed to produce the same fluorescein-type structure as had been previously prepared using fluorescein isothiocyanate (FITC). Our fluorescein phosphoramidite also contains a DMT group to allow quantification of coupling. The analogous structure, 6-Fluorescein Phosphoramidite, prepared using 6-FAM, is also available, along with 6-Fluorescein Serinol Phosphoramidite. Fluorescein-dT can be inserted into the desired sequence as a replacement for a dT residue.

We offer five fluorescein supports. Fluorescein CPG has traditionally been used 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 with 6-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 and use an amide linkage which is stable during cleavage and deprotection and does not allow isomer formation. 3'-(6-FAM) CPG allows effective blockage of the 3'-terminus from polymerase extension as well as exonuclease digestion. Fluorescein-dT CPG allows both of these enzymatic activities to proceed. Normal cleavage and deprotection with ammonium hydroxide readily generates the fluorescein labelled oligos. The spectral characteristics of these dyes are detailed on the following page.

The spectral characteristics of this dye is detailed here.

Details

Usage

  • Coupling: 3 minute coupling time recommended
  • Deprotection: Use ammonium hydroxide and deprotect as required by nucleobases. When using AMA, a small amount of a non-fluorescent impurity will be formed. To eliminate this impurity, first deprotect with ammonium hydroxide for 30 minutes at room temperature, add an equal volume of 40% methylamine and then complete the deprotection as required by the nucleobases - e.g. 10 minutes at 65°C or 2 hours at room temperature for standard bases.
Specifications
Diluent Anhydrous Acetonitrile
Storage Freezer storage, -10 to -30°C, dry
Stability 1-2 days, <90% efficient after 4 days


Dilution/Coupling Data

The table below show pack size data and, for solutions, dilution and approximate coupling based on normal priming procedures.

ABI 392/394

Catalog # Pack Size Grams/Pack 0.1M Dil. (mL) Approximate Number of Additions
LV40 LV200 40nm 0.2μm 1μm 10μm
10-5901-02 0.25 g .25grams 2.96 85.33 51.2 32 23.27 17.07 4.27
10-5901-90 100 µmol .084grams 1 20 12 7.5 5.45 4 1
10-5901-95 50 µmol .042grams 0.5 3.33 2 1.25 0.91 0.67 0.17

Expedite

Catalog # Pack Size Grams/Pack Dilution (mL) Approximate Number of Additions
Molarity 50nm 0.2μm 1μm 15μm
10-5901-02 0.25 g .25grams 4.42 0.07 82 51.25 37.27 5.13
10-5901-90 100 µmol .084grams 1.5 0.07 23.6 14.75 10.73 1.48
10-5901-95 50 µmol .042grams 0.75 0.07 8.6 5.38 3.91 0.54


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