Key featuresShow Hide
- Used to add a non-fluorescent quencher to the 5' end of an oligonucleotide.
- Maximal absorption in the 480 to 580 nm range.
- Does not have DMT functionality.
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.
- Chemical Name: 4'-(2-Nitro-4-toluyldiazo)-2'-methoxy-5'-methyl-azobenzene-4''-(N-ethyl)-N-ethyl-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite
- Formula: C34H45N8O5P
- Molecular Weight: 676.75
- Appearance: dark red solid
- Absorption Maximum (Lambda Max): 534
- Extinction Coefficient at Lambda max: 34000
- Extinction Coefficient at 260 nm: 8000
Spectral properties measured in PCR buffer as 5'-labeled poly(T) oligo.
- Dilution: 100 µmol/mL
- Deprotection conditions: Deprotection conditions are dependent upon the type of amidites used for the synthesis of the oligo. If using standard amidites deprotect in concentrated NH4OH for 5 hours at 60 °C. If using fast deprotecting amidites deprotect in concentrated NH4OH for 1 hour at 60 °C. If you are using base sensitive fluorophores (ie. TAMRA or Rhodamine based dyes) we recommend deprotecting the dye labeled oligonucleotide in t-butylamine/H2O (1:3) for 6 hours at 60 °C. If all the solution is not used during the synthesis, it can be stored under argon and its functionality maintained up to 48 hours.BHQ-1 amidite is susceptible to oxidation when left out in the open or undessicated.For detailed information on BHQ fragmentation by MALDI-TOF MS, refer to your BHQ Licensee's Manual Appendix V.Image of cleaved and deprotected structure:
- The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 538.49
Storage and handling:
- Shipping conditions: Cold
- Storage conditions: -15 to -30 °C