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Fluorescent Assays

qPCR Quenchers & Fluorophore Compatibility

Comprehensive dark quenchers and fluorescent acceptors mapped to FAM, HEX, Cy3, ROX, Cy5 — plus custom qPCR probe design & synthesis, dye–quencher pairing, multiplex panel design, and full analytical QC — scalable to ISO 13485 production.

Overview Dark Quenchers Fluorescent Quenchers Design Tips Related Services FAQ Speak to Scientist

Overview

electing the right quencher is critical for qPCR sensitivity, background suppression, and multiplex compatibility. Dark quenchers (non-emissive) such as BHQ-1/BHQ-2/BHQ-3, Iowa Black FQ/RQ, Dabcyl, QSY, and ATTO Quenchers efficiently absorb donor emission without adding stray fluorescence — ideal for TaqMan probes and molecular beacons. Fluorescent acceptors (e.g., TAMRA, ROX, Cy5) support FRET-based designs, calibration dyes, or specialized probe architectures.

Use this reference to match FAM, HEX/JOE/VIC, Cy3/TAMRA/ROX, Cy5/Cy5.5/Cy7 and other fluorophores with appropriate quenchers across blue–NIR channels, then apply the design tips below to optimize duplex stability, probe length, spacer placement, and instrument-specific filter sets.

Bio-Synthesis's services include custom qPCR probe design & synthesis (dual‑labeled TaqMan, molecular beacons, MGB options), dye/quencher pairing & multiplex panel design, and full analytical QC (HPLC, ESI‑MS; functional qPCR/ddPCR testing on request). We manufacture at scale from research grade to ISO 13485 production and offer related solutions such as custom DNA/RNA probes, diagnostic oligos for ELONA & lateral flow, enzyme‑labeled oligonucleotides, and biotinylated capture oligos for bead/plate workflows

Probe Design TaqMan & Beacons Multiplex Panels Analytical QC ISO 13485 qPCR / ddPCR

Services at a glance

  • Custom qPCR probes: dual‑labeled TaqMan, molecular beacons, MGB options
  • Dye/quencher pairing & multiplex panel design (FAM/HEX/ROX/Cy5 sets)
  • Analytical QC: HPLC, ESI‑MS; functional qPCR/ddPCR testing on request
  • Scale‑up & documentation: research to ISO 9001/13485 production
  • Related: enzyme‑labeled oligos, biotinylated capture oligos, diagnostic oligos (ELONA/lateral flow)

Dark Quenchers (Non-Emissive)

Pair BHQ-class, Iowa Black, Dabcyl, QSY, and ATTO Quenchers with common donors for TaqMan and beacon designs.

Hide Products and Notes
Quencher Abs Range (nm) Compatible Fluorophores Typical Usage Code
BHQ-0 ~430–480 FAM, TET, Fluorescein, CF488A, EvaGreen qPCR, SNP genotyping (blue/green dyes) [BHQ-0]
BHQ-1 ~480–580 FAM, HEX, JOE, VIC, Alexa 488, CF532 General qPCR, multiplexing [BHQ-1]
BHQ-2 ~560–670 Cy3, ROX, Alexa 555, TAMRA, CF568 Dual-labeled probes (orange-red dyes) [BHQ-2]
BHQ-3 ~620–730 Cy5, Alexa 647, Quasar 670, ATTO 633 Far-red qPCR, molecular beacons [BHQ-3]
Iowa Black FQ ~420–520 FAM, HEX, TET, Alexa 488 Alternative to BHQ-1, low autofluorescence [IBFQ]
Iowa Black RQ ~620–730 Cy5, Cy5.5, Alexa 647, Quasar 670, CF647 Far-red probes, long-wavelength FRET [IBRQ]
Dabcyl ~400–500 FAM, Fluorescein, TET, CF488A Classic quencher; ideal for green fluorophores [Dabcyl]
Eclipse ~400–650 FAM, Cy3, TET, ROX, HEX, Alexa 546 Broad-spectrum use, cost-effective [Eclipse]
QSY 7 ~470 Fluorescein, FAM Specialized for short-wavelength donors [QSY7]
QSY 21 ~650 Cy5, Alexa 647, ATTO 647, CF660 Red/NIR range detection [QSY21]
QSY 35 Broad Cy5, Cy7, ATTO 700, Alexa 750 Extended red to NIR range [QSY35]
QSY 9 ~650–700 Alexa 647, Quasar 670, ATTO 655 Red and far-red fluorophores [QSY9]
ATTO 540Q 525–600 FAM, HEX, Alexa 532, BODIPY FL Green/yellow-green fluorophores [ATTO540Q]
ATTO 580Q 550–610 Cy3, TAMRA, Alexa 555 Orange dyes, multiplexing [ATTO580Q]
ATTO 612Q up to ~670 Cy5, Alexa 633, CF647, ATTO 633 Far-red/near-IR dyes [ATTO612Q]
BBQ-650 ~550–750 Cy3, ROX, Alexa 555, Cy5, Alexa 647 Broadest coverage of commercial quenchers [BBQ650]
Epoch Eclipse Broad Spectrum FAM, Cy3, Cy5, ROX, HEX, TAMRA Versatile universal quencher [Epoch]
DYQ-1 Quencher 430–520 FAM, TET, HEX Green-yellow dyes; qPCR probes, dual-label FRET [DYQ1]
DYQ-2 Quencher 500–580 JOE, TAMRA, Alexa 532 Yellow-orange probes [DYQ2]
DYQ-4 Quencher 510–600 Cy3, ROX, Alexa 546 Orange-red dyes, molecular beacons [DYQ4]
DYQ-425 Quencher 520–620 TAMRA, ROX, CF555 TRITC-like dyes [DYQ425]
DYQ-505 Quencher 530–640 Cy3.5, CF568 Red dyes, dual-labeled FRET [DYQ505]
DYQ-660 Quencher 620–680 Cy5, Alexa 647, Quasar 670 Far-red dyes [DYQ660]
DYQ-661 Quencher 640–700 Cy5.5, CF660, ATTO 647N Red/NIR detection [DYQ661]
DYQ-700 Quencher 680–750 Cy7, Alexa 700, CF750 Near-IR quenching [DYQ700]
Technical Notes
  • Channel coverage: BHQ-1 (green), BHQ-2 (orange-red), BHQ-3 (far-red); Iowa Black FQ/RQ mirror green vs far-red ranges.
  • Background: Dark quenchers minimize bleed-through and simplify multiplexing in qPCR/ddPCR.
  • Probe architecture: 5′-reporter/3′-quencher TaqMan probes vs hairpin beacons may prefer different quenchers and spacer lengths.

Fluorescent Quenchers (Emit Light / FRET Acceptors)

Useful for FRET pairs, calibration dyes, or specialized designs that benefit from acceptor emission.

Show Products & Notes
Fluorescent Acceptor Abs (nm) Em (nm) Donor Fluorophores Typical Usage
TAMRA ~555 ~580 FAM, HEX, TET Classical acceptor; early dual-labeled probes
ROX ~585 ~605 JOE, TET, TAMRA qPCR calibration dyes; spectral separation
Cy5 ~649 ~670 Cy3, Alexa 546, TAMRA Far-red detection, long-range FRET
Alexa Fluor 594 ~590 ~617 Alexa 488, FAM, Oregon Green Photostable; FRET acceptor for multiplex
BODIPY 630/650-X ~630 ~650–665 Alexa 488, Oregon Green, FAM Green-to-red FRET acceptor
Texas Red® ~615 ~630–640 TAMRA, Cy3, Rhodamine, Alexa 546 FRET quencher for Cy3-class dyes
Technical Notes
  • FRET distance: Maintain appropriate donor–acceptor spacing (typically 2–6 nm) for efficient energy transfer.
  • Cross-talk: Acceptor emission can aid troubleshooting but adds a channel; verify instrument filters for multiplex.

Design Tips for qPCR Probes & Molecular Beacons

Choosing the Quencher

  • Match spectra: Pick a quencher whose absorption overlaps the donor’s emission peak.
  • Multiplexing: Prefer dark quenchers to minimize emission cross-talk across channels.
  • Probe length & Tm: Adjust length/spacers to maintain high on/off ratios; consider internal quenchers for short probes.

Assay Considerations

  • Instrument: Align donors with instrument filters (e.g., FAM/HEX/ROX/Cy5 standard sets).
  • Matrix effects: Detergents, dyes, and salts can shift background; pilot at working concentrations.
  • QC: Specify HPLC purification and confirm identity by MS; consider functional tests on request.

FAQ

When should I choose BHQ-1 vs BHQ-2 vs BHQ-3?

BHQ-1 pairs with green donors (FAM/HEX), BHQ-2 with orange-red (Cy3/ROX), and BHQ-3 with far-red (Cy5). Choose based on donor emission.

Do I need a fluorescent acceptor or a dark quencher?

Dark quenchers minimize background for multiplex qPCR. Fluorescent acceptors are useful for FRET and troubleshooting but add a detection channel.

How do spacers affect performance?

PEG/TEG spacers reduce sterics and can improve on/off ratios. Internal quenchers in short probes can further suppress background.

Any special considerations for ddPCR?

Use dark quenchers with tight spectral separation and verify droplet reader channels. Pilot dye/quencher combinations under final conditions.

Need help picking the best quencher for your dye set?

We’ll optimize donor/quencher pairs, probe length, and purification to match your instrument and multiplex design.

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