BHQ-2 CPG; Glycolate, Column
BHQ-2 CPG; Glycolate, Column
Key featuresShow Hide
- Used to add a non-fluorescent quencher to the 3' end of an oligonucleotide.
- Maximal absorption in the 560 to 670 nm range.
- Glycolate linker for rapid cleavage of the oligonucleotide.
- 500 Å CPG suitable for high yield applications (≤ 30mers).
- 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers).
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Since their introduction to the DNA marketplace in 2000, the Black Hole Quenchers have become THE gold-standard quencher of choice used in qPCR probes and other FRET applications. When fluorogenic dual-labeled probes were first introduced, quenchers were often a second reporter dye, typically TAMRA. The sensitivity of these probes, such as FAM-TAMRA, is limited because the fluorescence from TAMRA can leak into the channel meant to detect FAM fluorescence. Dark quenchers, which are dyes with no native fluorescence, offer a solution to this problem. The BHQ dyes are true dark quenchers with no native emission due to their polyaromatic-azo backbone. Substituting electron-donating and withdrawing groups on the aromatic rings produces a complete series of quenchers with broad absorption curves that span the visible spectrum into the near IR region. BHQ dyes work through a combination of FRET and static quenching to enable researchers to avoid the residual background signal common to fluorescing quenchers such as TAMRA, or low signal to noise ratio. These quenchers can be paired with all common reporter dyes to construct efficiently quenched qPCR probes for multiplexing assays. In addition to quenching by FRET, BHQ dyes have also been shown to efficiently quench fluorescence through static quenching via formation of a ground state complex with the reporter dye. BHQ quenchers have broad absorption spectra and can be paired with reporter dyes that emit in the following ranges: BHQ-0: 430-520 nm BHQ-1: 480-580 nm BHQ-2: 560-670 nm BHQ-3: 620-730 nm BHQ-10: 480-550 nm Water Soluble (WS) LGC, Biosearch Technologies offers all Black Hole Quencher products available for 3’, internal and 5’ modification of oligonucleotides with a variety of options. BHQ-1 and BHQ-2 are the more popular, either as the 5'-Phosphoramidites, the dT-Phosphoramidites or the 3'-CPGs. Only considering the excitation and emission values suggests Cy™5/Cyanine-5 and Quasar 670 require BHQ-3 for efficient quenching, however BHQ-2 is recommended because it is less susceptible to degradation. BHQ-1 is typically used to quench in the range 480 - 580 nm and can be used in conjunction with the commonly used fluorophores; e.g. FAM, TET, JOE and HEX. BHQ-2 is used to quench in the range 550 – 650 nm and is most effective in quenching fluorophores such as TAMRA, ROX, Cyanine-3, Cy3, Cy3.5™ and Red 640. We also offer Black Hole Quenchers for labelling peptides. All of our BHQ dyes are available as carboxylic acid or succinimidyl esters. Our BHQ-1 and BHQ-2 dyes are available as FMOC lysine conjugates. Our water soluble BHQ-10 is available as a carboxylic acid or succinimidyl ester.
- Appearance: purple powder
- Absorption Maximum (Lambda Max): 579
- Extinction Coefficient at Lambda max: 38000
- Extinction Coefficient at 260 nm: 8000
Spectral properties measured in PCR buffer as 3'-labeled poly(T) oligo.
- Cleavage conditions: When using fast deprotecting amidites use concentrated Ammonia for 45 min at 25 °C or Ammonia/ Methylamine (AMA) (1:1) for 15 min at 25 °C; or t-butylamine/H2O (1:3) for 90 min at 25 °C (TAMRA requires deprotection with TBA). When using standard amidites (eg. C-Bz, G-iBu) use concentrated Ammonia for 45 min at 25 °C.
- Deprotection conditions: When using fast deprotecting amidites (eg. C-Ac, G-DMF, G-PAC) deprotect in concentrated ammonia for 1 hour or AMA for 30 minutes at 60 °C. When using standard amidites (eg. C-Bz, G-iBu) deprotect in concentrated ammonia for 5 hours at 60 °C. If you are using base sensitive fluorophores (ie. TAMRA) we recommend deprotecting the oligonucleotide in t-butylamine/H2O(1:3) for 12-16 hours at 60 °C. ABI 3900 users: Columns should be compatible with standard ABI protocols. However, when using 50 nmol Super Columns, the following changes are recommended: 1) Change the head pressure/purge pressure/chamber pressure to 3.5 psi.2) An additional oxidation step3) OPTIONAL (but may help): extension of the coupling time to 20 secImage of cleaved and deprotected structure:
- The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 556.46
Storage and handling:
- Shipping conditions: Ambient
- Storage conditions: -15 to -30 °C in sealed container