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Oligo Synthesis

Oligo Synthesis : CEPs

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Fluorescein-dT Phosphoramidite

Fluorescein-dT Phosphoramidite

Glen Research

Catalogue No.DescriptionPack SizePriceQty
10-1056-02Fluorescein-dT Phosphoramidite 0.25g £614.00 Quantity Add to Order
10-1056-90Fluorescein-dT Phosphoramidite 100µmoles £368.00 Quantity Add to Order
10-1056-95Fluorescein-dT Phosphoramidite 50µmoles £193.00 Quantity Add to Order

Description

Fluorescein-dT Phosphoramidite

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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References

(1) B. Mullah and A. Andrus, Tetrahedron Lett, 1997, 38, 5751-5754.
(2) S.L. Woo, S.M. Menchen, and S. Fung, 1993, US Patent No. 5,231,191.

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Notes

Fluorescein-dT Phosphoramidite

5'-Dimethoxytrityloxy-5-[N-((3',6'-dipivaloylfluoresceinyl)-aminohexyl)-3-acrylimido]-
2'-deoxyUridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
Formula: C79H89N6O17P M.W.: 1425.57 F.W.: 815.71

Diluent: Anhydrous Acetonitrile
Coupling: 10 minute coupling time recommended.
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer.
Storage: Refrigerated storage, maximum of 2-8°C, dry
Stability in Solution: 24 hours
  Material Safety Data Sheet

Literature Highlights

Glen Report 10.1: NEW FLUORESCENT REAGENTS - TAMRA CPG, FLUORESCEIN-dT

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: Why does MALDI analysis of my oligos that contain one or more Fluorescein-dTs give an incorrect mass even though they give only a single, fluorescent band on a PAGE gel?

RESPONSE:It's an artifact of the MALDI analysis. The lasers used to ablate the MALDI matrix are generally between 308 and 355 nm, with the most frequently used laser line at 337 nm. When a laser hits a strong absorbance band of a molecule, often photochemistry starts to occur - which often leads to cleavage reactions. The Fluorescein-dT has strong UV absorbance in this region which appears to lead to a 135 m/z fragment is being blown off the molecule in a rather consistent manner. For instance, your oligos D1 and D2, which have 5 and 3 Flu-dTs respectively, the observed mass difference is -676 Da and -406 Da, which nicely fits the loss of a 135 mw fragment: 5 x 135 (675) and 3 x 135 (405). We haven't seen any papers that identify this 135 m/z fragment - but it's clear to me that that is what's occurring. Changing matrix matrix used may help. The matrix 2,4,6-trihydroxyacetophenone has been used successfully to analyze a fluorescein-labeled oligos[1], though the safest bet is to use Electrospray MS rather than MALDI for mass spec analysis.

REFERENCE(S):
1 Pieles et al., Nucleic Acids Res., Vol 21 (14) 3191-3196 (1993)


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
10-1056-02 0.25grams .25grams 1.75 45 27 16.88 12.27 9 2.25
10-1056-90 100µmoles .143grams 1 20 12 7.5 5.45 4 1
10-1056-95 50µmoles .071grams .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
10-1056-02 0.25grams .25grams 2.62 .067 46 28.75 20.91 2.88  
10-1056-90 100µmoles .143grams 1.5 .067 23.6 14.75 10.73 1.48  
10-1056-95 50µmoles .071grams .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
10-1056-02 0.25grams .25grams 2.62 .067 47.6 29.75 21.64    
10-1056-90 100µmoles .143grams 1.5 .067 25.2 15.75 11.45    
10-1056-95 50µmoles .071grams .75 .067 10.2 6.38 4.64    

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

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