Glen Research Product Information

Catalog Number: 10-1963-xx
Description: Fluorescein Phosphoramidite

1-Dimethoxytrityloxy-2-(N-thiourea-(di-O-pivaloyl-fluorescein)-4-aminobutyl)-propyl- 3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite

Formula: C68H79N4O12PS 
M.W.: 1207.50 
F.W.: 598.56 

Diluent: Anhydrous Acetonitrile

Coupling: 12-15 minute coupling time.

Deprotection: No changes needed from standard method recommended by synthesizer manufacturer.

Storage: Refrigerated storage, maximum of 2-8°C, dry

Stability in Solution: Unstable-Use same day as diluted

   

Pricing Information

Catalog Number

Pack Size

Price (US$)


10-1963-95
50µm
165.00
10-1963-90
100µm
295.00
10-1963-02
0.25g
595.00

Related Document(s)

  

Literature Highlight(s)

FREQUENTLY ASKED TECHNICAL QUESTION

QUESTION: Can oligonucleotides modified at the 5'-terminus with, for example, biotin be phosphorylated with kinase?

RESPONSE:Modification reagents based on a 1,2-diol, e.g., BioTEG, DNP phosphoramidites, or a 1,3-diol, e.g., fluorescein, biotin phosphoramidites, can be added several times at the 5'-terminus since they contain an alcohol group capable of further addition with phosphoramidites. Can this alcohol also be as a substrate for T4 polynucleotide kinase for 32P labelling of these modified oligonucleotides? Surprisingly, the answer is yes. Teoule and coworkers have shown(1) that oligos labelled at the 5'-terminus are substrates for kinase. Interestingly, the oligos modified with reagents based on 1,2-diols are labelled to 50%, indicating that only one diastereomer is labelled, while those modified with 1,3-diol reagents are labelled to 100%.

REFERENCE(S):
(1) M.L. Fontanel, H. Bazin, and R. Teoule, Analytical Biochemistry, 1993, 214, 338-340.


QUESTION: When quantifying a fluorescein labelled oligonucleotide by measuring absorbance at 260nm, what effect does the fluorescein have on this measurement?

RESPONSE:The extinction coefficient of DNA at 260nm is around 10,000/mole/base or 10/µmole/base. We use fluorescein isothiocyanate (Isomer 1) in the preparation of our fluorescein phosphoramidite (10-1963). The extinction coefficient of fluorescein (measured as FITC) at 260nm is around 13,700/mole or 13.7/µmole. The fluorescein contribution to the absorbance of a fluorescein labelled oligonucleotide at 260nm is, therefore, about the same as 1 base or about 5% of a 20mer. This may be neglected or corrected for in the determination of the amount of labelled oligonucleotide from an A260 measurement.

Note: dA=15.4, dC=7.4, dG=11.5, T=8.7

Fl-labeled ligos: 480nm excitation, 520nm emmission.

The extinction coefficient for fluorescein-5-isothiocyanate at 495 nm is 76,000 L/mole-cm at pH 9. Upon conjugation to protein, and by analogy oligo's, the extinction coefficient is decreased by 10%. This would result in a extinction coefficient of approximately 68,000 L/mole-cm. Additionally the extinction coefficient, and fluorescence emission, is very pH dependent (maximum at pH 9).

REFERENCE(S):
M. Powell, Glen Research


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: Can oligonucleotides modified at the 5'-terminus with, for example, biotin be phosphorylated with kinase?

RESPONSE:Modification reagents based on a 1,2-diol, e.g., BioTEG, DNP phosphoramidites, or a 1,3-diol, e.g., fluorescein, biotin phosphoramidites, can be added several times at the 5'-terminus since they contain an alcohol group capable of further addition with phosphoramidites. Can this alcohol also be as a substrate for T4 polynucleotide kinase for 32P labelling of these modified oligonucleotides? Surprisingly, the answer is yes. Teoule and coworkers have shown(1) that oligos labelled at the 5'-terminus are substrates for kinase. Interestingly, the oligos modified with reagents based on 1,2-diols are labelled to 50%, indicating that only one diastereomer is labelled, while those modified with 1,3-diol reagents are labelled to 100%.

REFERENCE(S):
(1) M.L. Fontanel, H. Bazin, and R. Teoule, Analytical Biochemistry, 1993, 214, 338-340.


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%


DILUTION/COUPLING DATA

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)
LV40LV20040nm0.2µm1µm10µm
Approximate Number of Additions
10-1963-020.25grams.25grams2.0755.6733.420.8815.1811.132.78
10-1963-90100µmoles.121grams120127.55.4541
10-1963-9550µmoles.06grams.53.3321.25.91.67.17
Expedite
Cat.No.Pack
Size
Grams/
Pack
Dilution
(mL)
Molarity50nm0.2µm1µm15µm
Approximate Number of Additions
10-1963-020.25grams.25grams3.09.0755.434.6325.183.46
10-1963-90100µmoles.121grams1.5.0723.614.7510.731.48
10-1963-9550µmoles.06grams.75.078.65.383.91.54

 

 

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01/21/2019

 

 

 

 

 

 

 

 

 

 

 

 

 

 


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