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CAL Fluor Red 590 Amidite

CAL Fluor Red 590 Amidite

A phosphoramidite used for 5'-CAL Fluor Red 590 labelling of oligonucleotides.
  • Used to add a yellow-orange fluorescent dye to the 5' end of an oligonucleotide.
  • Maximal emission around 590 nm. Alternative to TAMRA.
  • Quenched by BHQ-2.
  • Does not have DMT functionality.
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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.

Applicable Products

LK2423 3’-CAL Fluor® Orange 560 CPG 500
LK2424 3’-CAL Fluor® Red 610 CPG 500
LK2538 CAL Fluor® Orange 560-CE Phosphoramidite
LK2539 CAL Fluor® Red 590-CE Phosphoramidite
LK2540 CAL Fluor® Red 610-CE Phosphoramidite

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry, alcohol-free DCM or acetonitrile (see specifics below). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

LK2423 - - 1095.41 - - -
LK2424 - - 1171.44 - - -
LK2538 C40H53F6N5O4P2 843.83 560.60 2.96     5.93 11.85
LK2539 C42H57F6N5O4P2 871.88 588.65 2.87 5.73 11.47
LK2540 C46H57F6N5O4P2 919.93 636.69 2.72 5.44 10.87

Dissolution

LK2538 - Dilute to 0.1M in dry, alcohol-free DCM.

LK2539 & LK2540 - Dilute to 0.1M in dry acetonitrile.

In all cases the use of 4Å molecular sieves is highly recommended.

Coupling

A 15min coupling is recommended. Due to the mild deprotection conditions we recommend using fast deprotecting amidites. Prior to dilution of amidites ensure that all product is at the bottom of the vial. Dilute to the recommended concentration and mix thoroughly in sealed vial to ensure that all contents are dissolved. Once dissolved, add the sieves. We recommend using ETT (LK3140/2/5) as the activator for these products.

Cleavage & Deprotection

Cleave and deprotect the CAL dye labelled oligo in 2-methoxyethylamine/MeOH (1:3) for 3h at 60ºC (all products) or 6h at 25ºC (CPGs). Filter the sample and evaporate the supernatant. Re-dissolve the CAL dye labelled oligo in water. The use of strong bases, such as concentrated ammonia, causes degradation and must be avoided.

Storage & Stability

The products are light sensitive. Prolonged exposure to light may cause photo-bleaching. The products are also susceptible to oxidation when left out in the open or undesiccated. Store at -20ºC, desiccated. Amidite solutions should be stored under argon, but used within 24h.

Spectral Data

 

Absorbance Max./nm

Emission Max./nm

Fluorescent Region

Dye Replaces

CAL Fluor® Orange 560 537 558 yellow-orange VIC, TET, JOE
CAL Fluor® Red 590 569 591 yellow-orange TAMRA
CAL Fluor® Red 610 590 610 orange-red Texas Red®

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