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Oligo Synthesis

Oligo Synthesis : CEPs

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5'-Fluorescein Phosphoramidite

5'-Fluorescein Phosphoramidite

Glen Research

Catalogue No.DescriptionPack SizePriceQty
10-5901-025'-Fluorescein Phosphoramidite 0.25g £523.00 Quantity Add to Order
10-5901-905'-Fluorescein Phosphoramidite 100µmoles £195.00 Quantity Add to Order
10-5901-955'-Fluorescein Phosphoramidite 50µmoles £100.00 Quantity Add to Order

Description

  • 5'-Fluorescein Phosphoramidite

Structure

Catalog Number: 10-5901-xx

Description: 5'-Fluorescein Phosphoramidite

6-(3',6'-dipivaloylfluoresceinyl-6-carboxamido)-hexyl-
1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite
Formula: C46H58N3O10P M.W.: 843.95 F.W.: 537.46
Diluent: Anhydrous Acetonitrile
Coupling: 3 minute coupling time recommended
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer. Technical Bulletin
Storage: Freezer storage, -10 to -30°C, dry
Stability in Solution: 1-2 days, <90% efficient after 4 days
Please Note: This product or portions thereof is manufactured under license from GE Healthcare Bio-Sciences Corp. under US Patent Numbers 5,371,241, 5,721,355, and/or 5,583,236

FLUORESCEIN LABELLING

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-, hexachloro-and dichloro-dimethoxy-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.

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Protocols

MSDS

Glen Report 8.2

Glen Report 12.1

Glen Report 21.2

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Notes

5'-Fluorescein Phosphoramidite

6-(3',6'-dipivaloylfluoresceinyl-6-carboxamido)-hexyl-
1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite
Formula: C46H58N3O10P M.W.: 843.95 F.W.: 537.46
Diluent: Anhydrous Acetonitrile
Coupling: 3 minute coupling time recommended
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer. Technical Bulletin
Storage: Freezer storage, -10 to -30°C, dry
Stability in Solution: 1-2 days, <90% efficient after 4 days
Please Note: This product or portions thereof is manufactured under license from GE Healthcare Bio-Sciences Corp. under US Patent Numbers 5,371,241, 5,721,355, and/or 5,583,236
Store labeled oligo in the dark, either dry or in a neutral aqueous media at -20oC. Do not store crude fluorescently labeled oligonucleotides in ammonia.
Material Safety Data Sheet

Literature Highlights

Glen Report 8.2: 5' LABELLING - FLUORESCEIN AND CYANINE DYES, BIOTIN
Glen Report 12.1: POLY-PAK PURIFICATION OF LABELLED PROBES
Glen Report 21.2: Deprotection - Volume 3 - Dye-Containing Oligonucleotides

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.

DILUTION/COUPLING DATA

The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.

ABI 392/394
Cat.No. Pack
Size		 Grams/
Pack		 0.1M Dil.
(mL)		 LV40 LV200 40nm 0.2µm 1µm 10µm
Approximate Number of Additions
10-5901-02 0.25grams .25grams 2.96 85.33 51.2 32 23.27 17.07 4.27
10-5901-90 100µmoles .084grams 1 20 12 7.5 5.45 4 1
10-5901-95 50µmoles .042grams .5 3.33 2 1.25 .91 .67 .17
Expedite
Cat.No. Pack
Size		 Grams/
Pack		 Dilution
(mL)		 Molarity 50nm 0.2µm 1µm 15µm  
Approximate Number of Additions
10-5901-02 0.25grams .25grams 4.42 .067 82 51.25 37.27 5.13  
10-5901-90 100µmoles .084grams 1.5 .067 23.6 14.75 10.73 1.48  
10-5901-95 50µmoles .042grams .75 .067 8.6 5.38 3.91 .54  
Beckman
Cat.No. Pack
Size		 Grams/
Pack		 Dilution
(mL)		 Molarity 30nm 200nm 1000nm    
Approximate Number of Additions
10-5901-02 0.25grams .25grams 4.42 .067 83.6 52.25 38    
10-5901-90 100µmoles .084grams 1.5 .067 25.2 15.75 11.45    
10-5901-95 50µmoles .042grams .75 .067 10.2 6.38 4.64

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Applications & Benefits

DILUTION/COUPLING DATA

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

ABI 392/394
Cat.No. Pack
Size		 Grams/
Pack		 0.1M Dil.
(mL)		 LV40 LV200 40nm 0.2µm 1µm 10µm
Approximate Number of Additions
10-5901-02 0.25grams .25grams 2.96 85.33 51.2 32 23.27 17.07 4.27
10-5901-90 100µmoles .084grams 1 20 12 7.5 5.45 4 1
10-5901-95 50µmoles .042grams .5 3.33 2 1.25 .91 .67 .17
Expedite
Cat.No. Pack
Size		 Grams/
Pack		 Dilution
(mL)		 Molarity 50nm 0.2µm 1µm 15µm
Approximate Number of Additions
10-5901-02 0.25grams .25grams 4.42 .07 82 51.25 37.27 5.13
10-5901-90 100µmoles .084grams 1.5 .07 23.6 14.75 10.73 1.48
10-5901-95 50µmoles .042grams .75 .07 8.6 5.38 3.91 .54
Beckman
Cat.No. Pack
Size		 Grams/
Pack		 Dilution
(mL)		 Molarity 30nm 200nm 1000nm

Approximate Number of Additions
10-5901-02 0.25grams .25grams 4.42 .07 83.6 52.25 38

10-5901-90 100µmoles .084grams 1.5 .07 25.2 15.75 11.45

10-5901-95 50µmoles .042grams .75 .07 10.2 6.38 4.64

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Related products

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