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CAL Fluor Orange 560 Carboxylic Acid

CAL Fluor Orange 560 Carboxylic Acid

A carboxylic acid used for post-synthetic CAL Fluor Orange 560 labelling of oligonucleotides.
  • Used to add a yellow-orange fluorescent dye by conjugation to an amine functionality of a biomolecule.
  • Maximal emission around 560 nm. Alternative to VIC, HEX and JOE.
  • Quenched by BHQ-1.
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Product information

Historically, there was a limited selection of dyes available for multiplex PCR: TET, JOE, VIC™, HEX, Texas Red®, Cy™ 3, and Cy™ 5. In recent years, the selection and quality of reporters and quenchers has dramatically improved. LGC, Biosearch Technologies has developed the CAL Fluor Dyes, a set of fluorescent dyes specifically designed for qPCR instruments. These novel xanthene fluorophores not only replicate the applicability of previous dyes, they have further unique patented features. These dyes can be efficiently manufactured and remain stable through oligo synthesis and work up. The attachment chemistry linking the CAL Fluor Dyes to biomolecules eliminates the problem of multiple isomers. This results in dye labels that are easier to manufacture, have a single RP-HPLC peak and have well-defined emission spectra. CAL Fluor Dyes are available as CPGs and as phosphoramidites for oligonucleotide synthesis, which results in more efficient label incorporation and fewer purification steps than post-synthesis labeling. The CAL Fluor Dyes have emission maxima from 544 nm to 637 nm and can be paired with BHQ-1 or BHQ-2 for efficient quenching in a variety of probe formats. Multiplex suggestions and PCR instrument compatibility of CAL Fluor Dyes can be found here. Carboxylic acids of the CAL Fluor Dyes 560, 590, and 610 are available to use for peptide labelling.

Properties:

  • Formula: C31H36N3O4PF6
  • Molecular Weight: 659.6
  • Appearance: red solid
  • Absorption Maximum (Lambda Max): 533
  • Extinction Coefficient at Lambda max: 68000
  • Extinction Coefficient at 260 nm: 14000
  • Fluorescence Maximum: 556

Spectral properties measured in phosphate-buffered saline (PBS) pH 7.3.

Product usage:

    Product Solubility:This product can be dissolved in methanol, DMSO or DMF.Additional Spectroscopic Data:Absorption Maximum: 533 nm (PBS, pH 7.3); 534 nm (MeOH)Fluorescence Emission Maximum: 556 nm (PBS, pH 7.3), 556 nm (MeOH)e coefficient 534 nm = 89,000 cm-1M-1 (MeOH)e coefficient 533 nm = 68,000 cm-1M-1 (Water or PBS, pH 7.3)e coefficient 260 nm = 14,000 cm-1M-1 (Water or PBS, pH 7.3)e coefficient 280 nm = 11,600 cm-1M-1 (Water or PBS, pH 7.3)DIPCDI + HOAt Procedure for CAL Fluor Dye Addition to Peptide on Solid PhaseDIPCDI stock solution. 1 mL DIPCDI (di-isopropylcarbodiimide) + 9 mL DMF (HPLC quality).HOAt (1,2,3-Triazolo-[4,5-b]pyridin-3-ol, 1-hydroxyl-7-azabenzotriazole, TCI America T1673), 10 mg solid per 10 mg dye. Procedure. The peptide resin, 20mg, is placed in a syringe reactor (2ml polypro with coarse PE end frit), and the resin swollen in DMF. The dye (10mg) and HOAt (10mg) are placed in a screw capped 1.5 ml vial are treated with 110µL of freshly prepared DIPCDI solution, the vial sealed, and vortexed vigorously for 2 minutes. The dye solution is then diluted with DMF (200µL), mixed and admitted to the reactor. This is covered with foil and shaken overnight. The resin is washed 5x with DMF, treated 1x1 min with 30% piperidine in DMF, washed further with DMF (at least 5x) until no color in washes, washed 5x methanol, and all solvent expelled from syringe. Cleavage reagent (95%TFA/5% H20 made up under argon, 1.5 mL) is admitted to the syringe, which is shaken for 90 minutes. The TFA solution is then expelled into a 20mL glass scintillation vial and evaporated. The residue is dried in vacuo over KOH pellets. The product is analyzed by standard C-18 analytical HPLC in TFA/acetonitrile buffers with a PDA detector.Hazards This procedure, as do all peptide synthesis protocols, involves handling many dangerous, toxic, corrosive, irritant and poisonous chemicals. It should only be performed by a very experienced chemist in a well equipped laboratory fume hood with all possible caution. All waste should be disposed of according to safe laboratory procedures. Particular note should be made of the fact that HOAt is rated as being potentially explosive, it should be handled with care avoiding shock, heat or static electricity. Biosearch Technologies, Inc. assumes no responsibility for the safety of any part of this procedure, which has been supplied strictly for informational purposes only.HATU Procedure for CAL Fluor Dye Addition to Peptide on Solid PhaseDIPEA stock solution 1mL DIPEA (di-isopropylethylamine, glass distilled quality or better) + 9 mL DMF (HPLC quality).HATU [O-(7-Azabenzotriazolo-1-yl)-N,N.N',N'-tetramethyluronium PF6 salt, Aldrich 445460), 10 mg solid per 10 mg dye. Procedure. The peptide resin, 20mg, is placed in a syringe reactor (2 ml polypro with coarse PE end frit), and the resin swollen in DMF. The dye (10 mg) and HATU (10 mg) are placed in a screw capped 1.5 ml vial and treated with 110 µL of freshly prepared DIPEA solution, the vial sealed, and vortexed vigorously for 2 minutes. The dye solution is then diluted with DMF (200 µL), mixed and admitted to the reactor. This is covered with foil and shaken overnight. The resin is washed 5x with DMF, treated 1x1 min with 30% piperidine in DMF, washed further with DMF (at least 5x) until no color in washes, washed 5x methanol, and all solvent expelled from syringe. Cleavage reagent (95%TFA/5% H20 made up under argon, 1.5mL) is admitted to the syringe, which is shaken for 90 minutes. The TFA solution is then expelled into a 20mL glass scintillation vial and evaporated. The residue is dried in vacuo over KOH pellets. The product is analyzed by standard C-18 analytical HPLC in TFA/acetonitrile buffers with a PDA detector.Hazards This procedure involves handling many dangerous, toxic, corrosive and poisonous chemicals. It should only be performed by a very experienced chemist in a well equipped laboratory fume hood with all possible caution. All waste should be disposed of according to safe laboratory procedures. Particular note should be made of the fact that, although HATU is not rated as being potentially explosive, it is potentially just as hazardous as is HOAt, and should be handled with similar care, avoiding shock, heat or static electricity. Biosearch Technologies, Inc. assumes no responsibility for the safety of any part of this procedure, which has been supplied strictly for informational purposes only.

Storage and handling:

  • Shipping conditions: Ambient
  • Storage conditions: +2 to +8 °C

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