CAL Fluor Red 635 Amidite
CAL Fluor Red 635 Amidite
Key featuresShow Hide
- Used to add a orange-red fluorescent dye to the 5' end of an oligonucleotide.
- Maximal emission around 635 nm. Alternative to LC Red 640.
- Quenched by BHQ-2.
- Does not have DMT functionality.
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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.
- Formula: C50H63Cl2F6N5O4P2
- Molecular Weight: 1044.91
- Appearance: blue solid
- Absorption Maximum (Lambda Max): 616
- Extinction Coefficient at Lambda max: 112000
- Extinction Coefficient at 260 nm: 36500
- Fluorescence Maximum: 637
Spectral properties measured in PCR buffer as 5'-labeled poly(T) oligo.
- Dilution: 100 µmol/mL
- Deprotection conditions: For a 1 µmol to a 200 nmol synthesis scale, cleave and deprotect the CAL dye labeled oligo in 200 µL of 2-(2aminoethoxy)-ethanol (Aldrich # A54059) at 60 °C for 2.5 hours. The dye labeled CPG will lose the blue color after the addition of the 2-(2aminoethoxy)-ethanol. Remove the sample from the heat block and cool to approximately 25 °C. Add 1.5 mL of 1,4-dioxane. Shake the tube by hand. Centrifuge at 13,000 RPM for 30 seconds and remove the supernatant with a pipette. Repeat the process with another 1.5 mL of 1.4-dioxane. As the treatment with the 1,4-dioxane removes the base and precipitates out the DNA the sample should regain its blue color. After the removal of the 1,4-dioxane, add 1 ml of water containing 30 µL of acetic acid. CAL Fluor Red 635 will degrade if left in the presence of base for an extended amount of time.The use of strong bases, such as concentrated ammonia, causes degradation of the dye and should be avoided. CAL Fluor Red 635 is light sensitive. Prolonged exposure to light may cause photo bleaching.This product is susceptible to oxidation when left out in the open or undessicated. Unused amidite solution can be stored under argon and its functionality maintained for up to 24 hours.Image of cleaved and deprotected structure:
- The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 761.69
Storage and handling:
- Shipping conditions: Cold
- Storage conditions: -15 to -30 °C