Using Non-Fluorescent (Dark) Quenchers - BlackBerry® Quenchers

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Applicable Products

1378 BBQ-650®N-hydroxysuccinimide ester
2427 3'-BBQ-650® CPG II
2550 BBQ-650®-(DMT)-CE-Phosphoramidite

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions for 2550. 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

1378 C36H39N7O9 713.74 559.67 - - -
2427 - - 638.57 - - -
2550 C59H67N8O10P 1079.18 638.57 2.32 4.63 9.27

Oligo Synthesis with 2427 and 2550

Dissolution

2550 - This is sparingly soluble in acetonitrile therefore it is recommended that the amidite is solubilised in anhydrous DCM, AF (7 parts) and once completely dissolved (~10-15min) add anhydrous acetonitrile (3 parts). Do not premix the diluents. 0.1M concentration recommended.

Coupling

2550 – A 15min coupling time is recommended. Fast deprotection amidites are recommended.

After synthesis; it is recommended to flush the lines with DCM followed by MeCN to remove the dye immediately after use. This will prevent any precipitation or cross contamination.

2427 is 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. Fast deprotection amidites are recommended.

Cleavage & Deprotection

2550 -

  1. If the amidite is added to the 5’-end or the 3’-end on universal support. After synthesis, treat the support bound oligonucleotide with 20% DEA in acetonitrile (4028) to remove the cyanoethyl protection from the phosphate linkages. This will prevent any loss of the quencher during deprotection.
  2. Cleavage and deprotection are achieved in the same step by treating the support bound oligonucleotide with AMA, 10min, 65oC.

2427 -

  1. After synthesis, treat the support bound oligonucleotide with 20% DEA in acetonitrile (4028) to remove the cyanoethyl protection from the phosphate linkages. This will prevent any loss of the quencher during deprotection.
  2. Cleavage and deprotection are achieved in the same step by treating the support bound oligonucleotide with AMA, 10mins, 65oC.

Post-Synthetic Labelling with 1378

Solubility

DMF to 5-10mgmL-1 or 7-14µmolmL-1

Post synthetic labelling protocol

  1. Dissolve the amino functionalised biomolecule in 50mM carbonate buffer (pH 9.3).
  2. Dissolve the NHS ester in DMF.
  3. Add the solution from step 2 to the solution from step 1 such that the v:v of 1:2 is 2:1.
  4. Incubate overnight at 30oC.
  5. Quench the reaction with Tris-HCl buffer pH 7.2
  6. The conjugated product is now ready for purification.

Reference: A. Valanne, P. Malmi, H. Appelblom, P. Niemelä and T. Soukka, Anal. Biochem., 375, 71-81, 2008.

Spectral Data

ε (M-1cm-1) 40667 15077
λ 598nm 260nm
Solvent Methanol

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.

BlackBerry® Quencher technology: Covered under US Patent #7,879,986, Berry & Associates, Inc. BlackBerry Quencher,’ ‘BBQ 650 CEP,’  ‘BBQ 650 CPG’ and ‘BBQ 650 NHS Ester’ are trademarks of Berry & Associates, Inc., MI. The BBQ dye technology is sold under agreement with Berry & Associates, Inc. Products are sold exclusively for R&D use by the end user. They may not be used for clinical or diagnostic purposes.

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