Product info

Molecular beacon probes are dual-labelled probes that form a stem-loop (hairpin) structure, bringing the reporter and quencher into proximity. The loop region contains the sequence that hybridizes to the target sequence, while the complementary sequences at both ends of the probe form the stem. These probes generate fluorescence under non-hydrolytic conditions via hybridization to the target.

Molecular beacon structure:

  • 5′ fluorophore: The reporter dye emits fluorescence when the probe is linearised and hybridised to the target, separating the dye and quencher.
  • 3′ quencher: When the probe is in its hairpin structure, the proximity of the quencher to the reporter prevents fluorescence emission.
  • Stem: The stem is a double-stranded region formed by binding the complementary sequences (5-7 nt) at both ends of the probe.
  • Loop: The loop is a 18-30 nt sequence that is complementary to the target sequence.

 

Key benefits:

  • Enhanced specificity: The stem-loop conformation confers high specificity, making molecular beacons ideal for SNP/mismatch discrimination.
  • High signal-to-noise: Superior fluorophore quenching due to the proximity and the direct energy transfer between reporter and quencher while in the closed hairpin conformation.
  • Melt curve analysis suitability: Because molecular beacons generate fluorescence under non-hydrolytic conditions by target hybridisation, post-PCR melt curve analysis can be performed.
 

Select the best dye for your application

We offer molecular beacon probes labelled with a wide selection of dyes, enabling you to select options based on instrumentation and assay design. Quencher options for this probe type include Black Hole Quenchers (BHQ-1 and BHQ-2) and DABCYL dye.

5' fluorescent dye

Abs (nm)

Em (nm)

3' quencher

  FAM 495 520 BHQ-1, DABCYL
  TET 521 536 BHQ-1, DABCYL
  CAL Fluor Gold 540 522 544 BHQ-1, DABCYL
  CIV-550 530 550 BHQ-1, DABCYL
  JOE 529 555 BHQ-1, DABCYL
  HEX 535 556 BHQ-1, DABCYL
  CAL Fluor Orange 560 538 559 BHQ-1, DABCYL
  Quasar 570 548 566 BHQ-2, DABCYL
  Cy3 549 566 BHQ-2
  TAMRA 557 583 BHQ-2, DABCYL
  CAL Fluor Red 590 569 591 BHQ-2
  ROX 586 610 BHQ-2, DABCYL
  CAL Fluor Red 610 590 610 BHQ-2, DABCYL
  CAL Fluor Red 635 618 637 BHQ-2
  Cy5 646 669 BHQ-2
  Quasar 670 647 670 BHQ-2
  Quasar 705 690 705 BHQ-2

Use the spectral overlay tool to view compatible dyes for your instrument. Learn more

Order now

Molecular Beacon Probes usually ship in 8 business days.
  • Oligos for North America, South America, and APAC will be supplied out of our Petaluma, US manufacturing facility while oligos for EMEA will be supplied out of our Lystrup, Denmark manufacturing facility unless you have an agreement in place.
  • Our advertised turnaround time applies to orders received before 12PM for the local manufacturing site. Turnaround time is defined as the time of checkout until shipment.
Molecular Beacon Probes usually ship in 8 business days   More details

 

  • Oligos for North America, South America, and APAC will be supplied out of our Petaluma, US manufacturing facility while oligos for EMEA will be supplied out of our Lystrup, Denmark manufacturing facility unless you have an agreement in place.
  • Our advertised turnaround time applies to orders received before 12PM for the local manufacturing site. Turnaround time is defined as the time of checkout until shipment.

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Product listing

Catalog # Item name Price Note
MBO-C3B2-1
Molecular Beacon, 5' Cy3/3' BHQ-2 $1,017.00 Provides 40 nmol delivered.
MBO-C3B2-2
Molecular Beacon, 5' Cy3/3' BHQ-2 $724.50 Provides 10 nmol delivered.
MBO-C3B2-5
Molecular Beacon, 5' Cy3/3' BHQ-2 $646.50 Provides 3 nmol delivered.
MBO-FB1-1
Molecular Beacon, 5' FAM/3' BHQ-1 $837.00 Provides 50 nmol delivered.
MBO-FB1-2
Molecular Beacon, 5' FAM/3' BHQ-1 $592.00 Provides 25 nmol delivered.
MBO-FB1-5
Molecular Beacon, 5' FAM/3' BHQ-1 $523.00 Provides 5 nmol delivered.
MBO-FD-1
Molecular Beacon, 5' FAM/3' DABCYL $837.00 Provides 50 nmol delivered.
MBO-FD-2
Molecular Beacon, 5' FAM/3' DABCYL $592.00 Provides 25 nmol delivered.
MBO-FD-5
Molecular Beacon, 5' FAM/3' DABCYL $523.00 Provides 5 nmol delivered.
MBO-HB1-1
Molecular Beacon, 5' HEX/3' BHQ-1 $916.00 Provides 40 nmol delivered.
MBO-HB1-2
Molecular Beacon, 5' HEX/3' BHQ-1 $648.00 Provides 20 nmol delivered.
MBO-HB1-5
Molecular Beacon, 5' HEX/3' BHQ-1 $572.00 Provides 5 nmol delivered.
MBO-HD-1
Molecular Beacon, 5' HEX/3' DABCYL $916.00 Provides 40 nmol delivered.
MBO-HD-2
Molecular Beacon, 5' HEX/3' DABCYL $648.00 Provides 20 nmol delivered.
MBO-HD-5
Molecular Beacon, 5' HEX/3' DABCYL $572.00 Provides 5 nmol delivered.
MBO-JB1-1
Molecular Beacon, 5' JOE/3' BHQ-1 $993.00 Provides 40 nmol delivered.
MBO-JB1-2
Molecular Beacon, 5' JOE/3' BHQ-1 $703.00 Provides 15 nmol delivered.
MBO-JD-1
Molecular Beacon, 5' JOE/3' DABCYL $993.00 Provides 40 nmol delivered.
MBO-JD-2
Molecular Beacon, 5' JOE/3' DABCYL $703.00 Provides 15 nmol delivered.
MBO-RB2-1
Molecular Beacon, 5' ROX/3' BHQ-2 $993.00 Provides 40 nmol delivered.
MBO-RB2-2
Molecular Beacon, 5' ROX/3' BHQ-2 $703.00 Provides 15 nmol delivered.
MBO-RD-1
Molecular Beacon, 5' ROX/3' DABCYL $993.00 Provides 40 nmol delivered.
MBO-RD-2
Molecular Beacon, 5' ROX/3' DABCYL $703.00 Provides 15 nmol delivered.
MBO-TB2-1
Molecular Beacon, 5' TAMRA/3' BHQ-2 $873.00 Provides 40 nmol delivered.
MBO-TB2-2
Molecular Beacon, 5' TAMRA/3' BHQ-2 $612.00 Provides 20 nmol delivered.
MBO-TB2-5
Molecular Beacon, 5' TAMRA/3' BHQ-2 $544.00 Provides 5 nmol delivered.
MBO-TD-1
Molecular Beacon, 5' TAMRA/3' DABCYL $873.00 Provides 40 nmol delivered.
MBO-TD-2
Molecular Beacon, 5' TAMRA/3' DABCYL $612.00 Provides 20 nmol delivered.
MBO-TD-5
Molecular Beacon, 5' TAMRA/3' DABCYL $544.00 Provides 5 nmol delivered.
MBO-TEB1-1
Molecular Beacon, 5' TET/3' BHQ-1 $894.00 Provides 50 nmol delivered.
MBO-TEB1-2
Molecular Beacon, 5' TET/3' BHQ-1 $634.00 Provides 25 nmol delivered.
MBO-TEB1-5
Molecular Beacon, 5' TET/3' BHQ-1 $557.00 Provides 5 nmol delivered.
MBO-TED-1
Molecular Beacon, 5' TET/3' DABCYL $894.00 Provides 50 nmol delivered.
MBO-TED-2
Molecular Beacon, 5' TET/3' DABCYL $634.00 Provides 25 nmol delivered.
MBO-TED-5
Molecular Beacon, 5' TET/3' DABCYL $557.00 Provides 5 nmol delivered.
MBO-CAB2-2
Molecular Beacon, 5' CAL Fluor Red 610/3' BHQ-2 $655.00 Provides 20 nmol delivered.
MBO-CAB2-5
Molecular Beacon, 5' CAL Fluor Red 610/3' BHQ-2 $578.00 Provides 5 nmol delivered.
MBO-CAB2-1
Molecular Beacon, 5' CAL Fluor Red 610/3' BHQ-2 $922.00 Provides 40 nmol delivered.
MBO-CAD-5
Molecular Beacon, 5' CAL Fluor Red 610/3' DABCYL $578.00 Provides 5 nmol delivered.
MBO-CAD-2
Molecular Beacon, 5' CAL Fluor Red 610/3' DABCYL $655.00 Provides 20 nmol delivered.
MBO-CAD-1
Molecular Beacon, 5' CAL Fluor Red 610/3' DABCYL $922.00 Provides 40 nmol delivered.
MBO-C5B2-5
Molecular Beacon, 5' Cy5/3' BHQ-2 $646.50 Provides 3 nmol delivered.
MBO-C5B2-2
Molecular Beacon, 5' Cy5/3' BHQ-2 $724.50 Provides 10 nmol delivered.
MBO-C5B2-1
Molecular Beacon, 5' Cy5/3' BHQ-2 $1,017.00 Provides 30 nmol delivered.
MBO-Q5B2-5
Molecular Beacon, 5' Quasar 570/3' BHQ-2 $572.00 Provides 5 nmol delivered.
MBO-Q5B2-2
Molecular Beacon, 5' Quasar 570/3' BHQ-2 $648.00 Provides 25 nmol delivered.
MBO-Q5B2-1
Molecular Beacon, 5' Quasar 570/3' BHQ-2 $916.00 Provides 50 nmol delivered.
MBO-Q6B2-5
Molecular Beacon, 5' Quasar 670/3' BHQ-2 $578.00 Provides 5 nmol delivered.
MBO-Q6B2-2
Molecular Beacon, 5' Quasar 670/3' BHQ-2 $655.00 Provides 20 nmol delivered.
MBO-Q6B2-1
Molecular Beacon, 5' Quasar 670/3' BHQ-2 $922.00 Provides 40 nmol delivered.
MBO-COB1-1
Molecular Beacon, 5' CAL Fluor Orange 560/3' BHQ-1 $873.00 Provides 40 nmol delivered.
MBO-COB1-2
Molecular Beacon, 5' CAL Fluor Orange 560/3' BHQ-1 $612.00 Provides 20 nmol delivered.
MBO-COB1-5
Molecular Beacon, 5' CAL Fluor Orange 560/3' BHQ-1 $544.00 Provides 5 nmol delivered.
MBO-COD-1
Molecular Beacon, 5' CAL Fluor Orange 560/3' DABCYL $873.00 Provides 40 nmol delivered.
MBO-COD-2
Molecular Beacon, 5' CAL Fluor Orange 560/3' DABCYL $612.00 Provides 20 nmol delivered.
MBO-COD-5
Molecular Beacon, 5' CAL Fluor Orange 560/3' DABCYL $544.00 Provides 5 nmol delivered.
MBO-C590B2-5
Molecular Beacon, 5' CAL Fluor Red 590/3' BHQ-2 $530.00 Provides 5 nmol delivered.
MBO-C590B2-2
Molecular Beacon, 5' CAL Fluor Red 590/3' BHQ-2 $597.00 Provides 20 nmol delivered.
MBO-C590B2-1
Molecular Beacon, 5' CAL Fluor Red 590/3' BHQ-2 $858.00 Provides 40 nmol delivered.
MBO-C635B2-2
Molecular Beacon, 5' CAL Fluor Red 635/3' BHQ-2 $718.00 Provides 20 nmol delivered.
MBO-C635B2-1
Molecular Beacon, 5' CAL Fluor Red 635/3' BHQ-2 $922.00 Provides 40 nmol delivered.
MBO-C635B2-5
Molecular Beacon, 5' CAL Fluor Red 635/3' BHQ-2 $578.00 Provides 5 nmol delivered.
MBO-CGD-5
Molecular Beacon, 5' CAL Fluor Gold 540/3' DABCYL $544.00 Provides 5 nmol delivered.
MBO-CGD-2
Molecular Beacon, 5' CAL Fluor Gold 540/3' DABCYL $612.00 Provides 20 nmol delivered.
MBO-CGD-1
Molecular Beacon, 5' CAL Fluor Gold 540/3' DABCYL $873.00 Provides 40 nmol delivered.
MBO-CGB1-5
Molecular Beacon, 5' CAL Fluor Gold 540/3' BHQ-1 $544.00 Provides 5 nmol delivered.
MBO-CGB1-2
Molecular Beacon, 5' CAL Fluor Gold 540/3' BHQ-1 $612.00 Provides 20 nmol delivered.
MBO-CGB1-1
Molecular Beacon, 5' CAL Fluor Gold 540/3' BHQ-1 $873.00 Provides 40 nmol delivered.
MBO-Q5D-1
Molecular Beacon, 5' Quasar 570/3' DABCYL $916.00 Provides 50 nmol delivered.
MBO-Q5D-5
Molecular Beacon, 5' Quasar 570/3' DABCYL $572.00 Provides 5 nmol delivered.
MBO-Q5D-2
Molecular Beacon, 5' Quasar 570/3' DABCYL $648.00 Provides 25 nmol delivered.
MBO-Q7B2-5
Molecular Beacon, 5' Quasar 705/3' BHQ-2 $578.00 Provides 5 nmol delivered.
MBO-Q7B2-2
Molecular Beacon, 5' Quasar 705/3' BHQ-2 $655.00 Provides 20 nmol delivered.
MBO-Q7B2-1
Molecular Beacon, 5' Quasar 705/3' BHQ-2 $922.00 Provides 40 nmol delivered.
MBO-CIB1-1
Molecular Beacon, 5' CIV/3' BHQ-1 $873.00 Provides 40 nmol delivered.
MBO-CIB1-2
Molecular Beacon, 5' CIV/3' BHQ-1 $612.00 Provides 20 nmols delivered.
MBO-CIB1-5
Molecular Beacon, 5' CIV/3' BHQ-1 $544.00 Provides 5 nmol delivered.
MBO-CID-1
Molecular Beacon, 5' CIV/3' DABCYL $873.00 Provides 40 nmols delivered.
MBO-CID-2
Molecular Beacon, 5' CIV/3' DABCYL $612.00 Provides 20 nmols delivered.
MBO-CID-5
Molecular Beacon, 5' CIV/3' DABCYL $544.00 Provides 5 nmols delivered.

Technical Specs

Molecular Beacon Probe specifications
Dyes FAM, TET, CAL Fluor gold 540, CIV-550, JOE, HEX, CAL Fluor Orange 560, Quasar 570, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, CAL Fluor Red 635, Cy5, Cy3 Quasar 670, Quasar 705
Quenchers BHQ-1, BHQ-2, DABCYL
Yield Dye dependent (see product listing)
Purification DUAL HPLC
Delivery format Dry or in solution (water, TrisHCl, T10E0.1, T10E1)
Quality control MS and UHPLC
Quality standard ISO 9001 or ISO 13485
Shelf life 12 months from date of manufacture

 

FAQs

  • Product Usage:

    • Can I use the Nanodrop® to measure the concentration of synthetic oligonucleotides?

      Nanodrop® technology can be used to measure the concentration of individual synthetic oligos using each oligonucleotide's unique analysis constant. By default, the Nanodrop equipment uses a value of "33" as a general constant for all single-stranded DNA, which is inappropriate for synthetic DNA. Oligonucleotides purchased through LGC Biosearch Technologies arrive with data sheets containing the extinction coefficient and molecular weight of each oligonucleotide. These numbers are used to calculate the analysis constant needed for Nanodrop concentration calculations.

      Use the formula below to calculate the Analysis Constant (AC):
      AC = (1/extinction coefficient) x (Molecular Weight (protonated)) x 1000 = AC in micrograms per OD260nm

      We have determined through internal research that when measuring labeled oligonucleotides, the Nanodrop's linear range of detection is much more limited than advertised. For oligonucleotide stocks in the 100 µM range, the Nanodrop will record an apparent concentration that is significantly below the actual concentration. For accurate measurements, we recommend diluting 100 µM stocks by 25-fold to achieve a concentration in the range of 4 µM.

    • How do you determine the brightness of a dye?

      The absolute intensity of a dye is a product of the extinction coefficient and the quantum yield. We have not measured the quantum yield for our dyes as this value is highly dependent upon the local environment, including the buffer system used for the measurement. However, we do provide the extinction coefficients for dye modifications at their lambda max wavelength, and these values are available under the Technical Specs tabs of our Oligo Modifications webpages. While quantum yield and extinction coefficients both contribute to dye detectability, the principal determinant for Stellaris® RNA FISH assays is actually the instrument optics, including the excitation source, available filters, and quantum efficiency of the camera.

    • How do I quantify oligonucleotides by spectrophotometer?

      Here is a protocol for the Quantification of Oligonucleotides by Spectrophotometer:

      1. Add an aliquot of the resuspended oligonucleotide into a volume of PBS so that the total volume is 1000 µl. Typical dilutions are 1:20 or 1:40 where the dilution factor (DF) is 1000/aliquot volume.
      2. Vortex or pipette up and down repeatedly for 15 seconds.
      3. Read the absorbance of this dilution at 260 nm (OD260). Use the average of at least 2 reads.
      4. Calculate concentration using the nmol/OD260 value presented on the Certificate of Analysis, i.e. multiply (nmol/OD260) x (average OD260) x (Dilution Factor) = [C], concentration in µM (micromolarity).
    • How do I calibrate my instrument for the CAL Fluor® and Quasar® Dyes?

      CAL Fluor® and Quasar® dye calibration standards are designed to improve the accuracy of signal detection in real-time thermal cyclers that require spectral calibration. They enable the instrument to store the fluorescence profile of each dye and control for channel cross-talk. Crosstalk is the bleed-through of fluorescent signal from a reporter into an adjacent filter or channel, an issue of particular concern in a multiplexed assay. Many qPCR machines are pre-calibrated for Cy™3 and Cy5 dyes. In those machines, no calibration is necessary to use our Quasar 570 (Cy3 alternative) and Quasar 670 (Cy5 alternative) dyes. To use our CAL Fluor dye labels, particularly in a multiplexing assay, certain real-time PCR instruments need to be calibrated to anticipate crosstalk. LGC Biosearch Technologies does not make available pure dyes. Instead, our calibration standards are formulated to better mimic a fluorescent probe under experimental conditions by covalently linking the dye to an oligo-thymidine (dT10).  A complete list of available Calibration and Reference Dyes is available through our website. Instructions to calibrate select qPCR machines are available in our Spectral Calibration Instructions.

    • How many PCR reactions will I be able to run with my probe or primer?

      The number of reactions per nmol of product delivered is dependent upon the concentration to be used and final reaction volume. Typically, 1 nmol of a primer designed for qPCR will provide sufficient material for at least 100 reactions if used at a 300 nM final concentration in a 20 µL total volume. Likewise, 1 nmol of dual-labeled BHQ probe will provide sufficient material for up to 500 reactions if used at a 100 nM final concentration in a 20 µL total volume.

    • How do I adjust my thermal cycler’s settings to account for the BHQ® quencher?

      Dual-labeled BHQ®, BHQplus®, or BHQnova™ probes may be used on any qPCR instrument. These probes exhibit extremely low background fluorescence, enhancing detection sensitivity. The selection process for the quencher dye during set-up varies between instruments. Because Black Hole Quencher® dyes have no fluorescence emission, simply choose the setting for 'Non-fluorescent', 'dark quencher' or ‘none’.

  • qPCR:

    • What is the difference between static quenching and FRET?

      The static quenching mechanism is the formation of an intramolecular dimer between reporter and quencher, to create a non-fluorescent ground-state complex with a unique absorption spectrum. In contrast, the FRET quenching mechanism is dynamic and does not affect the probe's absorption spectrum. With either mechanism, disruption of quenching through hydrolysis of the probe releases signal from the fluorophore.

      For more information, please visit our Quenching Mechanisms in Probes webpage.

    • Which dyes are compatible with my thermal cycler?

      LGC Biosearch Technologies offers many common fluorophores including FAM, HEX and TAMRA dyes, as well as our own proprietary dyes. Our CAL Fluor® and Quasar® dye series span the spectrum with emission wavelengths ranging from yellow to far-red, and represent alternatives to dyes such as VIC®, Cy™3, Texas Red, LC Red® 640, Cy5, and Cy5.5. For your convenience we have compiled a Multiplexing Dye Recommendations Chart outlining optimal dye combinations in select qPCR machines, as well as a Fluorophore & BHQ® Dye Selection Chart listing reporter-quencher pairings. In addition, you may use our Spectral Overlay Tool to visualize the absorption and emission spectra of multiple dyes together.

    • Does LGC Biosearch Technologies make available information on multiplex qPCR?

      For information on qPCR assay design, validation and troubleshooting please visit our Multiplexing qPCR webpage and review the information under the tabs. Additional information is presented in our blog series, The BiosearchTech Blog. If you have further questions, please contact our Technical Support team.

  • Building Probes/Primers:

    • What different oligonucleotide purification options does LGC Biosearch Technologies offer?

      LGC Biosearch Technologies offers a full range of purification options including: Salt-free, Reverse Phase Cartridge (RPC), Reverse Phase HPLC (RP-HPLC), Anion Exchange HPLC (AX-HPLC) and Dual-HPLC (AX-HPLC followed by RP-HPLC). They are listed from least to most stringent, with the appropriate purification depending entirely on the application.

      For unlabeled oligonucleotides, such as qPCR primers, Salt-free or RPC purification is appropriate. For other applications using unmodified oligonucleotides we encourage RPC purification which typically provides ~70 % purity. With RPC purification, contaminants such as truncated sequences, ammonium salts and impurities are removed from the final product. In this process, the oligos are synthesized with the DMT group left on the final base which allows for separation by affinity of the DMT group to the resin in the cartridge. Truncated sequences will not have the final DMT group, will not bind to the cartridge and will be washed away.

      RP-HPLC is selected to eliminate fluorescent contaminants that remain following synthesis of a labeled oligo. When allowed to persist, this impurity elevates the baseline fluorescence and obscures the detection of probe signal. RP-HPLC typically yields products with ~80 % purity. This purification technique is similar to RPC purification except the resins provide greater sample capacity.

      AX-HPLC is selected to eliminate failure sequences that result from poor reporter or base coupling during the synthesis. When allowed to persist, this impurity competes with the oligo for binding to the target sequence which may result in delayed CT values in a qPCR reaction.

      For Dual-labeled BHQ® probes we recommend at a minimum RP-HPLC purification, but default to Dual-HPLC which typically provides products with ~90 % purity.

      In oligonucleotides containing wobbles, we avoid AX-HPLC which skews the ratio of different species synthesized in unison.

      For more information, please review our Default and Recommended Methods of Purification Chart.

    • What is the difference between Black Hole Quencher® (BHQ®) dye and TAMRA?

      TAMRA dye is an effective quencher for fluorophores with emission maxima less than 560 nm. Dyes with longer wavelength emissions will not be effectively quenched by TAMRA. In addition, TAMRA has its own fluorescence which complicates data analysis due to crosstalk between the channels. In contrast, Black Hole Quencher® dyes are true "dark" quenchers with no fluorescent signal. Their use simplifies design, implementation and interpretation of qPCR assays.

      Furthermore, BHQ® dyes have broad absorption spanning 480-580 nm (BHQ-1), 559-670 nm (BHQ-2) and 620-730 nm (BHQ-3), to enable use of a large range of spectrally distinct reporter dyes in multiplexed assay designs. With some dye pairings, FRET quenching is supplemented by the static quenching mechanism. Specifically, hydrophobic and electrostatic interactions facilitate the association of BHQ dyes with certain reporters to form an intramolecular dimer, for enhanced quenching and improved signal to noise ratios. Thus, BHQ dyes may quench some fluorophores whose emission spectrum is beyond the limits of BHQ absorption. For more information on FRET and static quenching mechanisms in qPCR please visit our Quenching Mechanisms in Probes website.

    • What is the difference between LGC Biosearch Technologies' Quasar® dyes and the Cy™ dyes?

      The Quasar® dyes may be used as direct replacements for the Cy™ dyes and are anticipated to perform equivalently to their Cy dye counterparts. They share the same chromophore structure and spectral properties, differing principally in their linkage chemistry. Quasar 570 replaces Cy3, Quasar 670 replaces Cy5 and Quasar 705 replaces Cy5.5 dye. Quasar dyes are slightly more hydrophobic and therefore soluble in the reagents of DNA synthesis. Importantly, the Quasar dyes are available as amidites and may be incorporated during oligonucleotide synthesis, thus avoiding the post-synthesis dye conjugation required with Cyanine dyes.

    • What are "wobbles"?

      When comparing multiple sequences, one may find that alignment reveals no region with sufficient consensus to accommodate a unique single oligonucleotide for use as a primer or probe. In some cases, only one or two nucleotides are mismatched. When designing primers for those regions, one may choose to introduce a degenerate site, or "wobble", to compensate for the variability in the target sequence. Letter codes are used to represent the combination of two or more different nucleotide phosphoramidites blended at equimolar ratios prior to coupling at that position in the sequence. The final product is a blend of two or more different sequences made simultaneously during one synthesis.

      2 nucleotide wobble
      R = A+G
      W = A+T
      M = A+C
      Y = C+T
      S = C+G
      K = G+T

      3 nucleotide wobble
      B = C+T+G
      D = A+G+T
      H = A+C+T
      V = A+C+G

      Universal wobble
      N = A+C+T+G
    • Does LGC Biosearch Technologies offer VIC®, NED or PET dyes?

      LGC Biosearch Technologies does not offer VIC®, NED or PET dyes as they are proprietary to Applied Biosystems, Inc. (part of Life Technologies). These dyes are often used for sequencing or fragment analysis, but other long-wavelength dyes do not perform well in fragment analyzers, such as the ABI 3730 series.  These types of instruments use a single wavelength (488 nm) for excitation which poorly excites red-shifted dyes. Applied Biosystems circumvents this problem by partnering red dyes such as NED with a FAM dye in a FRET construct. LGC Biosearch does not offer these “Big Dye” constructs and so we advise testing our dyes on an experimental basis for fragment analysis.

      For qPCR applications we do not offer direct replacements for NED or PET dyes, however, we do offer alternatives for VIC.  Our recommended VIC substitute depends on the optics of your qPCR machine which can be determined on our Multiplexing Dye Recommendations Chart

    • What Black Hole Quencher® do you recommend for dyes with long wavelength emissions, such as the Quasar® and Pulsar® dyes?

      The BHQ®-2 dye is our preferred quencher for long wavelength fluorophores. This recommendation relates to the ease of manufacture using BHQ-2 over BHQ-3 dye. While both dyes represent excellent quenchers, the final yield is usually higher with BHQ-2 modified oligonucleotides, thus providing a more cost-effective synthesis with excellent purity and performance characteristics.

      In the context of Dual-labeled BHQ probes, the BHQ-2 dye is an excellent quencher for long wavelength emitters such as Quasar® 670, Quasar 705, and Pulsar® 650. With some dye pairings, FRET quenching is supplemented by the static quenching mechanism. Specifically, hydrophobic and electrostatic interactions facilitate the association of BHQ dyes with certain reporters to form an intramolecular dimer, for enhanced quenching and improved signal to noise ratios. Thus, BHQ dyes may quench some fluorophores whose emission spectrum is beyond the limits of BHQ absorption. More information on FRET and static quenching can be found on our Quenching Mechanisms in Probes webpage.

    • Do unlabeled primers have a 3' phosphate?

      Unless otherwise requested at the time of the order, unlabeled primers are synthesized with free hydroxyls at both the 5' and 3' ends. Terminal phosphate modifications are available as custom modifications only.

      For a list of available modifications and associated pricing, please visit our Oligo Modifications webpage or contact our Customer Service team.

    • Where can I find information that explains the differences between each of the probe types you offer?

      We offer a number of different qPCR probe types for your convenience, including: Dual-labeled BHQ® probes, BHQnova™ probes, BHQplus® probes, Molecular Beacons, and Scorpions® Primers. For detailed information about how these probes work, please watch our Real-time PCR Probe Animation Video. You may also download our Fluorogenic Probes and Primers Brochure.

  • Troubleshooting:

    • I am performing real time qPCR and my negative controls are amplifying. Why would this happen?

      The most common reason negative controls come up positive is cross-contamination by a positive control such as a plasmid template. Below are a few suggestions to prevent contamination:

      1) Aliquot your probe and primers into small aliquots with enough product to run only a few experiments. Not only does this guard against contamination but will also help minimize the number of freeze/thaw cycles which degrade oligonucleotide quality.

      2) Use separate work areas for qPCR reagent preparation, DNA/template addition and amplification product handling.

      3) Clean qPCR work areas and pipettes (designated for qPCR use only) regularly with a DNA degradative agent and follow up with 70% ethanol.

      4) Use only sterile, filtered pipette tips to minimize aerosol contamination of the pipettes.

      5) If you continue to have trouble, consider using Uracil-N-Glycosylase (UNG) in your assay set up.
    • Why do I get different Ct values for the same probe sequence when labeled with a different dye?

      It is not unusual to observe slightly different cycles to threshold (Ct or Cq) values for the same probe sequence labeled with different fluorophores. Such variation is typically on the order of 1 to 2 cycles and relates to the differences in dye intensity as well as the variation in instrument optics across the different channels. Fundamentally, all real-time thermal cyclers are engineered to detect fluorescein (FAM) first and foremost, so dyes with longer wavelength emission may be detected less sensitively. Occasionally, changing the fluorophore can have a profound impact on functional performance, particularly when the melting temperature of the probe is marginal. Such an outcome might relate to the hydrophobic attraction between modifications, or a change in melting temperature with the new fluorophore.
    • Why is it that when a sequence contains a ‘wobble’ it has variable functionality?

      Incorporation of 'wobbles' into a sequence decreases the effective concentration of each species. With increased numbers of 'wobbles' the number of distinct species increases exponentially thereby decreasing the likelihood that any individual sequence has the desired specificity. Only one species is usually present in a biological sample. As some portion of the oligo species are not completely complementary to the target, some variability in function is to be expected. To further complicate matters, the individual nucleotide amidites can have different coupling rates. Each time the same 'wobble' sequence is synthesized, there is the potential that one species will be produced in preference over another.

      Tips:
      1) Be conservative. Introduce as few 'wobbles' as possible; one trinucleotide 'wobble' and one dinucleotide 'wobble' or two dinucleotide 'wobbles' in two different locations such that there is a maximum of 6 variants in a single oligonucleotide and;

      2) Increase the concentration of your 'wobble' sequence by as much as two fold to compensate for the presence of multiple unique sequences.

      If you have specific questions regarding minimum yields for a particular probe, please contact our Technical Support team.
  • Product Documentation:

    • Why does the synthesis scale ordered for my oligonucleotide not correspond with the final yield?

      The synthesis scale represents the quantity of precursor used to begin the manufacture of the oligonucleotide. Because the synthesis cycle proceeds with a finite efficiency some material is lost while coupling each nucleotide monomer, as well as cleavage of the oligonucleotide from the solid support, and finally preparative purification following synthesis. The aggregate loss from each of these steps cause the final yield of the oligonucleotide to be less than the starting synthesis scale.
    • Does LGC Biosearch Technologies make Safety Data Sheets (SDS) available?

      Safety Data Sheets (SDS), formerly referred to as Material Safety Data Sheets (MSDS), are available for download under the Technical Specs or Related Info tabs found on most product webpages. If you are unable to access this document or need additional information, please contact our Technical Support team.

    • Does LGC Biosearch Technologies measure A260/A280 ratios of the oligonucleotides?

      Measuring the Absorbance at 260nm / 280nm ratio is not appropriate when applied to synthetic DNA. The A260/A280 ratio is used to measure the purity of DNA following applications in which protein contamination may be an issue, such as DNA extraction from cells. As protein contamination is not a concern with synthetic DNA, we do not use this ratio.

      See reference: Validity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratio. Biotechniques 18(1): p 62-63, 1995.
    • Where can I find usage information and chemical properties for Biosearch Technologies' products?

      Product information sheets contain important usage information and chemical properties. You may download the product information sheets, by clicking on the appropriate link located under the Technical Specs tab of individual product pages. If for any reason you are unable to find what you need on our website, please e-mail our Technical Support team.