Using Non-Fluorescent (Dark) Quenchers - Black Hole Quenchers

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Applicable Products

2154 5'-BHQ-1™-CE Phosphoramidite
2155 5'-BHQ-2™-CE Phosphoramidite
2156 BHQ-1™-dT-CE Phosphoramidite
2157 BHQ-2™-dT-CE Phosphoramidite
2379 3'-BHQ-1™ CPG
2380 3'-BHQ-1™ CPG

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry, alcohol-free dichloromethane for 2156 & 2157, and dry acetonitrile (4050) for 2154 & 2155. 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

2154 C34H45N8O5P 676.76 538.49 3.69 7.39 14.78
2155 C33H43N8O6P 678.73 540.47 3.68 7.37 14.73
2156 C74H89N12O14P 1401.56 960.93 1.78 3.57 7.13
2157 C73H87N12O15P 1403.53 962.91 1.78 3.56 7.12
2379 - - 554.49 - - -
2380 - - 556.47 - - -

Dissolution

The dT amidites (2156 & 2157) are dissolved in anhydrous (alcohol-free) dichloromethane, and the 5’-amidites (2154 & 2155) are dissolved in anhydrous acetonitrile, all to standard concentrations. Prepare these amidite solutions at least 10min before placing on the synthesiser.

Coupling

2154, 2155 & 2156– A 10min coupling time is recommended.

2157 – A 15min coupling time is recommended.

The CPGs are used per standard solid supports following the synthesiser instructions. Non-nucleosidic CPG supports do not detritylate as rapidly as nucleosidic ones, therefore an additional detritylation step is recommended. It is therefore necessary to use a cycle that does not contain an initial capping step.

Cleavage & Deprotection

When selecting a deprotection method for oligos containing the BHQ™ phosphoramidites, use conditions suitable for any other modifications on the oligonucleotide. The BHQ™ component itself is stable to typical standard, UltraFAST or UltraMILD conditions. When using a base-sensitive fluorophore such as TAMRA, UltraMILD deprotection (0.05M potassium carbonate in methanol for 4h at room temperature) or deprotection with tbutylamine/water (1:3) for 2.5h at 70°C is recommended.

The CPGs have a glycolate linkage. This allows for rapid cleavage of the oligonucleotides and is labile enough for base-sensitive oligos. For cleavage use ammonium hydroxide for 20min at 25°C or AMA for 5min at 25°C; or, when using TAMRA, tbutylamine/methanol/water (1:1:2) for 45min at 25°C.

Complete the deprotection with conditions suitable for the other nucleobases or modifications as above.

Spectral Data

 

Absorbance Max./nm

BHQ-1™ 534
BHQ-2™ 579

Analysis

Note that if analysing oligos after purification by MS, an additional peak at M-298 (BHQ-1) or M-300 (BHQ-2) is observed. This is attributed to fragmentation within the MS through the diazo bond.

Storage & Stability

Products are stored in light-protected containers in the freezer at –10 to –30°C. All phosphoramidites are susceptible to oxidation when left exposed to air and/or moisture. All phosphoramidites are stable in solution under argon for 2 days.

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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