Cyanine Labelling

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Applicable Products

2158 Quasar™ 570-CE Phosphoramidite
2159 Quasar™ 670-CE Phosphoramidite
2412 3'-Cyanine-3 SynBase™ CPG 1000/110 (Cyanine 540)
2413 3'-Cyanine-5 SynBase™ CPG 1000/110 (Cyanine 650)
2520 Cyanine-3-CE Phosphoramidite (Cyanine 540)
2521 Cyanine-5-CE Phosphoramidite (Cyanine 650)

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (4050) with the exception of 2521 where anhydrous alcohol free DCM is required. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight. We suggest also using 3-4Å activated molecular sieves (10% v/v).

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

2158 C44H65N5O4P 759.00 622.74 3.29 6.59 13.18
2159 C46H67N5O4P 758.04 648.78 3.18 6.37 12.74
2412 - - 507.59 - - -
2413 - - 633.63 - - -
2520 C58H70ClN4O4P 953.64 507.59 2.62 5.24 10.49
2521 C60H72ClN4O4P 979.68 533.63 2.55 5.10 10.21

Coupling

A 10min coupling time is recommended for all phosphoramidites. Due to the milder conditions required when deprotecting cyanine-labelled oligonucleotides, the use of fast or ultramild deprotection amidites (Ac-dC, Bz-dA, dmf-dG, iPrPac-dG, Pac-dA) is recommended. The use of 4A molecular seives is recommended.

Deprotection

As these reagents require no deprotection themselves, the oligonucleotide deprotection strategy employed is guided by the protective groups on the rest of the oligo. However, the mildest possible conditions are recommended, especially for Quasar™ 670 (2159) and Cyanine 5 (2521) which are susceptible to degradation at elevated temperatures.

Cleavage and deprotection: Do this in one step. Remove the resin from the column and place into a deprotection tube. Add ammonium hydroxide and allow to stand for 2-3hr at room temperature. The deprotection solution must be removed immediately after deprotection (see below).

Biosearch recommend deprotection of the Quasar dyes with ammonium hydroxide for 8hr at 25°C or for 1 hr at 60°C, however we have obtained good quality oligos with the above conditions, especially as elevated temperatures increase degradation.

Removing the deprotection solution: This must be done as soon as the deprotection step is complete. It is important not to heat the oligo to remove the deprotection solution since this will degrade the dye. Pass the solution through a G25 (NAP10 or equivalent) column. This will remove the basic solution and the sample is ready for purification. Some users pass the solution through two columns to be absolutely sure that the solution is now ~ neutral pH.

Spectral Data

 

Absorbance Max./nm

Emission Max./nm

Colour

Quasar™ 570 547 570 Yellow-Orange
Quasar™ 670 644 670 Red
Cyanine 3 546 563 Yellow-Red
Cyanine 5 646 662 Violet-Far Red

Storage & Stability

These products are light-sensitive and are susceptible to oxidation when left exposed to air and/or moisture. Store dry in a freezer, in a sealed dark or amber container at –20°C. Similarly, modified oligonucleotides must also be protected from exposure to light. The phosphoramidites are stable in solution under argon for 24h.

Black Hole Quencher, BHQ, CAL Fluor and Quasar, are registered trademarks of Biosearch Technologies, Inc. (BTI). All BTI products offered for sale by LINK are sold under the condition that they be used for research purposes only and are prohibited from use in diagnostic or other applications unless explicitly authorized by separate written agreement with BTI. Black Hole Quencher® (BHQ®) CAL Fluor®, and Quasar® dyes (referred to collectively as "BTI Dyes") are sold to the purchaser for internal R&D use only and are not to be used for clinical or clinical diagnostic purposes. Neither the BTI Dyes nor the compounds synthesized with them are to be re-packaged or re-sold. Separate licenses for other than the aforementioned internal R&D applications of the BTI Dyes may be available. Please inquire via info@biosearchtech.com. The Black Hole Quencher® dye technology is protected in the United States and other countries by U.S. patents and continuations numbered 7,019,129, 7,019,129B1, 7,109,312B2, 7,582,432, 8,410,255B2 and 8,440,399B2 issued to Biosearch Technologies, Inc. The CAL Fluor® technology is covered by U.S. patent number 7,344,701B2. The Quasar® technology is covered by U.S. Patent numbers 7,705,150B2, 7,868, 157B2 and 8,436,153B2.

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