Which synthesis column will work best with my machine?

To assist you in selecting the correct column, please refer to our downloadable Column Selection Guide.

I am getting low coupling efficiency with amidites. Do you have any suggestions on how to improve it?

What is the difference between LGC Biosearch Technologies' Quasar® dyes and the Cyâ„¢ 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.

Which one of your Black Hole Quencher® dyes do you recommend using to label proteins?

Our Black Hole Quencher® 10 succinimidyl ester (BHQ-10S), is a water soluble BHQ® dye specifically engineered for conjugating to proteins. BHQ-10 will function as a FRET quencher for fluorophores located up to 100 angstroms away. All other BHQ products are insoluble in aqueous solutions and are not appropriate for protein labeling.

How stable are the CAL Fluor® and BHQ® dyes when conjugated to peptides in solution?

Does the LGC Biosearch Technologies' website have a list of all dyes available for DNA labeling?

LGC Biosearch Technologies offers modified oligos with many common fluorophores including FAM, HEX and TAMRA, as well as our own proprietary dyes. Please refer to our Black Hole Quencher® and Dye Selection Chart for a complete list of various dyes/fluorophores we carry at LGC Biosearch.

LGC Biosearch makes available many of these same dyes as reactive precursors for others to synthesize their own modified oligos. A full list of dyes and quenchers formulated for that purpose can be found in on our webpage for DNA/RNA Synthesis Reagents.

To manually label oligos and other biomolecules, LGC Biosearch also offers carboxylic acid and succinimidyl ester formulations of certain dyes and quenchers. A complete list can be found on our Labeling Reagents webpage.

If you have any questions regarding the availability of particular products, please contact our Technical Support team.

Does LGC Biosearch Technologies recommend the use of FAST deprotecting or standard amidites?

LGC Biosearch Technologies recommends the use of FAST deprotection amidites such as: Adenosine-benzoyl,A(Bz;) Cytidine-acetyl,C(Ac); Guanosine-dimethylformamide, G(Dmf). For ultra-FAST deprotection, use the phenoxyacetyl (Pac) amidites: A (Pac), C (Pac) and G (Pac). Thymidine does not need a protecting group. The FAST deprotecting amidites are used when dye-labeled oligonucleotides are expected to be sensitive to basic conditions, especially when long time periods of incubation are involved. This is true for the CAL Fluor® dyes which are known to degrade in concentrated NH₄OH.

The use of a tetrazole activator is in concurrence with our chemistry practices. LGC Biosearch uses ethylthioltetrazol (ETT) for all amidite productions. Be advised that the 4,5-dicyanoimidizole (DCi) activator, while less acidic, more soluble and more nucleophilic than tetrazol or ETT, does NOT work with our CAL Fluor chemistries.

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. 

Are LGC Biosearch Technologies' dyes compatible with DNA sequencing or fragment analyzers?

LGC Biosearch Technologies' dyes have not been tested for compatibility in DNA sequencers or instruments for fragment analysis. While the potential is there, many DNA sequencers are engineered for use with “Big Dye” FRET constructs, such as FAM/NED, to compensate for the limited excitation provided by the laser. LGC Biosearch does not offer these constructs. We encourage users to test our dyes in their instrument prior to designing an assay.

LGC Biosearch dyes are principally intended for use in probe-based studies such as qPCR and SNP genotyping.

What formulations of BHQ® dyes do you offer for internal modifications of oligonucleotides?

We make available two formulations of both the BHQ®-1 dye and BHQ-2 dye for use as internal modifications. BHQ dyes may be incorporated as either an abasic formulation or else attached to a deoxythymidine (dT) nucleoside. The abasic formulation of the modifications will disrupt the continuity of the sugar-phosphate backbone and may impact oligonucleotide geometry upon hybridization. When ordering, indicate abasic BHQ internal modifications by inserting [BHQ-1] or [BHQ-2] within your oligo sequence. The T-BHQ formulation is recommended when the sugar-phosphate linkage must be preserved. When ordering, indicate this modification using [T(BHQ-1)] or [T(BHQ-2)] within your oligo sequence. Here are examples showing proper demarcations: ACGT[T(BHQ-1)]ACGT for the T-linked formulation, or ACGT[BHQ-1]ACGT for the abasic modification.

For more information, see our Blog article named 'Labeling Oligos with Internal BHQ dyes'.

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 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.

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.

Which conjugation ratio (loading) should I use for an ELISA?

How do I know which loading to choose for immunochemical conjugates?

Are LGC Biosearch Technologies' immunochemical products alum precipitated?

Our immunochemical products are not alum precipitated and we do not offer this service. You may find a protocol for alum precipitation in the following reference:

Adjuvant effect of bacterial LPS and/or alum precipitation in response to polysaccharide and protein antigens. Seppälä IJ, Mäkelä O. Immunology 1984 Dec, 53(4):827-836  PMCID: PMC1454889"

How do I calculate the conjugation ratios (loading) for immunochemicals?

Conjugation ratio is the molar ratio of hapten to protein. We recommend using Beer’s Law to calculate the conjugation ratio:
A = ε * L * C
Below is an example conjugation ratio calculation for NP-BSA (4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Bovine Serum Albumin).

Known values
A_NP (Measured absorbance of the hapten (NP) at 430 nm, pH 8.5): for this example we will use OD430 = 1
ε_NP (Extinction coefficient of the hapten (NP) at 430 nm): 4230 M^-1 cm^-1
L (Path length):  typically this value is assumed to be 1 cm

Calculate C_NP (Molar concentration of the hapten (NP))
C_NP  = A_NP / (ε_NP * L)
C_NP  = OD430 (no units) / (ε_NP (M^-1 cm^-1) * 1 cm) 
C_NP  = 1 / 4230 M^-1
C_NP  = 0.000236 Molar or 236 uM

Calculate C_BSA (Molar concentration of BSA)
If provided a mass concentration, make sure to convert to molar concentration by dividing the mass concentration by molecular weight.  For example, if NP-BSA is dissolved in 0.1 M NaHCO₃ (pH 8.5) to 1 mg / mL (as recommended per the use instructions found under the Technical Specs tab of the product page ), then BSA has a mass concentration of 1 mg / mL or 1 g / liter.

Knowing that BSA has an approximate molecular weight (MW) of 60,000 g / mol, the molar concentration of BSA is then:
C_BSA = Mass concentration / MW
C_BSA = (1 g / liter) / (60,000 g / mol)
C_BSA = 0.0000167 mol / liter or 16.7 uM

Calculate conjugation ratio (molar ratio (n) of the hapten (NP) to (BSA))
n = C_NP/C_BSA
n = 236 uM / 16.7 uM
n ≈ 14 NP haptens conjugated to each BSA molecule (NP14-BSA)

Can I order a specific conjugation ratio for my immunochemical?

LGC Biosearch Technologies does not offer custom immunochemical productions. The conjugation ratio for any given immunochemical will vary with each production. We do not have a means of anticipating the ratio of the next production. We do not anticipate changes in performance with small modifications in molar ratio, such as 1 to 2 haptens per protein.  Available conjugation ratios may be found under the ‘Loadings’ pull down menu, located to the right of the ‘Quantity‘ box, at the bottom of each product-specific webpage within the Immunochemicals product category.

Does the LGC Biosearch Technologies' website have a list of all dyes available for DNA labeling?

LGC Biosearch Technologies offers modified oligos with many common fluorophores including FAM, HEX and TAMRA, as well as our own proprietary dyes. Please refer to our Black Hole Quencher® and Dye Selection Chart for a complete list of various dyes/fluorophores we carry at LGC Biosearch.

LGC Biosearch makes available many of these same dyes as reactive precursors for others to synthesize their own modified oligos. A full list of dyes and quenchers formulated for that purpose can be found in on our webpage for DNA/RNA Synthesis Reagents.

To manually label oligos and other biomolecules, LGC Biosearch also offers carboxylic acid and succinimidyl ester formulations of certain dyes and quenchers. A complete list can be found on our Labeling Reagents webpage.

If you have any questions regarding the availability of particular products, please contact our Technical Support team.

What are "wobbles"?

Why is it that when a sequence contains a ‘wobble’ it has variable functionality?

I have ordered a 5' thiol C6 modification on my custom oligonucleotide. What form does that come in and how do I create a free thiol?

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 proper notation if I want to use a deoxyinosine within my oligonucleotide sequence?

Does LGC Biosearch Technologies have a list of all available oligonucleotide modifications on its website?

At LGC Biosearch Technologies we use the phosphoramidite method of oligonucleotide synthesis. This method allows us to offer a broad selection of fluorescent and non-fluorescent modifications for 5', 3' and internal labeling. For a complete listing of available modifications, please review our Oligo Modifications webpages.

If you have any questions about our modifications, please contact our Technical Support team.

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.

Are locked nucleic acids (LNAâ„¢) available through LGC Biosearch Technologies?

LGC Biosearch Technologies is not licensed to synthesize oligos with locked nucleic acid (LNA™) modifications.

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. 

Are LGC Biosearch Technologies' dyes compatible with DNA sequencing or fragment analyzers?

LGC Biosearch Technologies' dyes have not been tested for compatibility in DNA sequencers or instruments for fragment analysis. While the potential is there, many DNA sequencers are engineered for use with “Big Dye” FRET constructs, such as FAM/NED, to compensate for the limited excitation provided by the laser. LGC Biosearch does not offer these constructs. We encourage users to test our dyes in their instrument prior to designing an assay.

LGC Biosearch dyes are principally intended for use in probe-based studies such as qPCR and SNP genotyping.

What formulations of BHQ® dyes do you offer for internal modifications of oligonucleotides?

We make available two formulations of both the BHQ®-1 dye and BHQ-2 dye for use as internal modifications. BHQ dyes may be incorporated as either an abasic formulation or else attached to a deoxythymidine (dT) nucleoside. The abasic formulation of the modifications will disrupt the continuity of the sugar-phosphate backbone and may impact oligonucleotide geometry upon hybridization. When ordering, indicate abasic BHQ internal modifications by inserting [BHQ-1] or [BHQ-2] within your oligo sequence. The T-BHQ formulation is recommended when the sugar-phosphate linkage must be preserved. When ordering, indicate this modification using [T(BHQ-1)] or [T(BHQ-2)] within your oligo sequence. Here are examples showing proper demarcations: ACGT[T(BHQ-1)]ACGT for the T-linked formulation, or ACGT[BHQ-1]ACGT for the abasic modification.

For more information, see our Blog article named 'Labeling Oligos with Internal BHQ dyes'.

If I order a dual-labeled probe with an internal BHQ® modification, what should be at the 3' end of the probe?

For Dual-labeled BHQ® probes that contain an internal BHQ modification, you should specify an additional Spacer 3 (C3) modification at the 3' terminus, to prevent extension of the probe.

You may want to consider our BHQnova™ Probes instead of adding an internal BHQ modification to your probe. BHQnova probes are a double-quenched probe format that improves quenching efficiency without impacting signal release, for improved signal-to-noise ratios. Additionally, BHQnova probes are a more economical option than probes with an internal T(BHQ-1) and Spacer C3 modification.

BHQnova probes incorporate our internal “Nova” quencher between base residues 9 & 10 from the 5’ end in addition to the 3’ terminal BHQ modification. This quencher configuration is well suited to probe sequences 25 bases or longer which otherwise may suffer from poor quenching efficiency as traditional end-labeled probes.

What are the recommended storage conditions for Dual-labeled BHQ® probes?

How are fluorescent labels on Dual-labeled BHQ® probes accounted for when measuring the absorbance?

Extinction coefficients are a prediction of each oligo’s molar absorbance and the standard method to calculate concentration. To estimate the extinction coefficient of modified oligonucleotides, we use the following equation: ε260 = [(Sum of ε260 for all bases) + (ε260 for all modifications)] x 0.9, to adjust for hyperchromicity. Individual extinction coefficients for each modification are available under the Technical Specs tabs on our Oligo Modifications webpages. The extinction coefficient found on the Certificate of Analysis for a custom oligo includes all dye and other modifications in the presented value. Simply use this value and measure the OD at 260 nm to calculate oligo concentration according to Beer’s Law. 

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 determine the volume of buffer to add to my probe to prepare a stock concentration?

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.

What formulations of BHQ® dyes do you offer for internal modifications of oligonucleotides?

We make available two formulations of both the BHQ®-1 dye and BHQ-2 dye for use as internal modifications. BHQ dyes may be incorporated as either an abasic formulation or else attached to a deoxythymidine (dT) nucleoside. The abasic formulation of the modifications will disrupt the continuity of the sugar-phosphate backbone and may impact oligonucleotide geometry upon hybridization. When ordering, indicate abasic BHQ internal modifications by inserting [BHQ-1] or [BHQ-2] within your oligo sequence. The T-BHQ formulation is recommended when the sugar-phosphate linkage must be preserved. When ordering, indicate this modification using [T(BHQ-1)] or [T(BHQ-2)] within your oligo sequence. Here are examples showing proper demarcations: ACGT[T(BHQ-1)]ACGT for the T-linked formulation, or ACGT[BHQ-1]ACGT for the abasic modification.

For more information, see our Blog article named 'Labeling Oligos with Internal BHQ dyes'.

How do I know what fluorophore to pick for my Dual-labeled BHQ® probe?

The choice of fluorescent reporter to label your dual-labeled BHQ® probe(s) depends upon your instrument optics and also the degree of multiplexing you want to achieve. If your assays will be amplified separately then we encourage you to label each probe with FAM. FAM is the most commonly used fluorophore and is detected by all real-time PCR instruments. The optic capabilities of the instrument, i.e. excitation source and filters, determine the degree of multiplexing and which fluorophores can be used. For a listing of our available dyes, download our Black Hole Quencher® and Dye Selection Chart. You may find information on multiplexing and a table listing our recommended dye choices for a selection of qPCR machines on our Multiplexing qPCR webpage.

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.

Does LGC Biosearch Technologies synthesize minor groove binder (MGB) probes?

Minor groove binder (MGB) probes are proprietary to Life Technologies (part of Thermo Fisher) and unavailable through LGC Biosearch Technologies. Instead, we offer an alternative chemistry called BHQplus^®, but BHQplus probes do not represent a direct replacement for MGB probes. Nevertheless, BHQplus represents a duplex stabilizing chemistry to allow for shorter sequences with elevated melting temperatures than their sequences would indicate. BHQplus probes are specially designed for SNP genotyping, and to detect more difficult targets such as AT-rich targets or short conserved regions.

For more information, please visit our BHQplus Probes webpage.

What is the difference between BHQ® and BHQplus® Probes?

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.

If I order a dual-labeled probe with an internal BHQ® modification, what should be at the 3' end of the probe?

For Dual-labeled BHQ® probes that contain an internal BHQ modification, you should specify an additional Spacer 3 (C3) modification at the 3' terminus, to prevent extension of the probe.

You may want to consider our BHQnova™ Probes instead of adding an internal BHQ modification to your probe. BHQnova probes are a double-quenched probe format that improves quenching efficiency without impacting signal release, for improved signal-to-noise ratios. Additionally, BHQnova probes are a more economical option than probes with an internal T(BHQ-1) and Spacer C3 modification.

BHQnova probes incorporate our internal “Nova” quencher between base residues 9 & 10 from the 5’ end in addition to the 3’ terminal BHQ modification. This quencher configuration is well suited to probe sequences 25 bases or longer which otherwise may suffer from poor quenching efficiency as traditional end-labeled probes.

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.

I am a beginner at real-time qPCR. Does LGC Biosearch Technologies have information which will help me to design my assay?

For an overview of available Dual-labeled BHQ® probe types, their mode of action and basic design guidelines, you may download our Fluorogenic Probes and Primers Brochure.

For an in depth discussion on qPCR, including probe and primer design, we recommend reading the book entitled 'A-Z of Quantitative PCR' edited by Stephen A. Bustin.

Additional resources are available on-line, including the website 'REAL-TIME PCR' maintained by M. Tevfik Dorak, MD, Ph.D., which offers a review of major topics for qPCR and historical links to valued information.

For MIQE guidelines on experiment design, please see the original publication entitled, "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments", by Bustin et al.

For applications and the most recent methods in gene expression analysis, visit the website named 'www.Gene-Quantification.info: The Reference in qPCR - Academic & Industrial Information Platform'. This site offers many links to additional resources on qPCR.

Does LGC Biosearch Technologies have software available for designing probes and primers?

For the design of qPCR primers and probe sets, we offer our RealTimeDesign™ (RTD™) software. The RTD software is free, easy to use and can be accessed directly through your web browser. Our software offers a choice of design modes: an 'Express Mode' with pre-set parameters and a 'Custom Mode' in which the parameters can be adjusted by the user. There is an additional 'Batch Mode' which facilitates the design of up to 10 assays in series. 

Is there a formula for calculating the efficiency of a qPCR reaction?

For a singleplex reaction, the efficiency of qPCR is calculated as follows:

Efficiency = 10^(-1/slope) - 1

The slope is derived from a graph of Cycles to Threshold (Ct) values plotted against the Log₁₀ of the template amount. A slope of -3.32 indicates an amplification efficiency of 100%.

Resource: Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. David G. Ginzinger. Experimental Hematology 30 (2002): 503-512

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 the FAM-BHQ ValuProbeâ„¢ and the small sized FAM-BHQ probe?

The difference is in the delivery amount and the purification method used. A The difference is in the delivery amount and the purification method used. A ValuProbe™ BHQ® Probe (Cat# DLO-RFB-5) delivers exactly 10 nmol of material, while the small scale FAM-BHQ-1 probe (Cat# DLO-FB1-5) has a minimum delivery of 10 nmol but averages closer to 20 nmol. ValuProbe BHQ Probes are purified using reverse phase HPLC only, whereas the small scale probe is purified by dual-HPLC (anion exchange followed by reverse phase HPLC). Dual-HPLC purification ensures the highest quality of dual-labeled BHQ probes and is recommended for standard probe sequences that will be ordered repeatedly. For more detailed information, please review our Purification Options webpage or contact our Technical Support team.

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.

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.

Are locked nucleic acids (LNAâ„¢) available through LGC Biosearch Technologies?

LGC Biosearch Technologies is not licensed to synthesize oligos with locked nucleic acid (LNA™) modifications.

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. 

Why do the sequences for my BHQplus® Probes appear different than before?

Do I need to calibrate my instrument to detect LGC Biosearch Technologies' Pulsar® 650 dye?

The Pulsar® 650 dye is appropriate for use in the Roche LightCycler® 1.2 and 2.0 qPCR machines. The use of a two color (FAM and Pulsar 650 dye) duplex assay requires spectral calibration of the machine. Spectral calibration will decrease cross-talk between channels. This is achieved by a color compensation (CC) file loaded into your LightCycler computer. Dual-labeled BHQ® probe users have the flexibility to create this file in one of three ways:

1. Download a previously-saved CC file according to your instrument model
2. Generate an assay-specific CC file using the Dual-labeled BHQ probes directly or
3. Generate a universal CC file using T10 calibration dyes.

Unfortunately, Molecular Beacons cannot be used directly to generate a CC file.

For instructions on how to create CC files, please download our Methods of Color Compensation on the LightCycler® Instruments_FAM/Pulsar 650 Duplexed Assays guide.

Color Compensation Files:

• LightCycler™ 1.2 Color Compensation File
• LightCycler™ 2.0 Color Compensation File

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’.

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.

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.

I am having a problem with background drift with my Dual-labeled BHQ® probe. What might the cause be?

I am not getting any signal generation with my Dual-labeled BHQ® probe. What might cause this?

De novo assay designs often require optimization and no signal generation may be an indication of inappropriate oligo design. However, failure to amplify may also be due to an oversight in the reaction preparation, particularly if the assay has performed well in the past. To pinpoint the problem component, review the topics below:

Tip: Try to avoid designing a probe with a Guanosine at the 5' end as G may quench some of the fluorophore emission and decrease signal generation.

1. Were all the MasterMix components added appropriately? If repeated, does the assay continue to fail?
2. Are the sequences of the probes and primers on the tube labels and Certificate of Analysis correct? Are the fluorophore and quencher modifications correct?
3. When you analyze the primer set with SYBR Green chemistry or run the failed qPCR reaction products on an agarose gel, do you get the correct amplification product (T_M and size)? If so, then reviewing the probe sequence may help.  Consider the following factors in your current probe design:
   • Probes longer than 30 bases have insufficient quenching and are frequently non-functional. This length limitation of probe design can be overcome with the use of our Double Quenched BHQnova™ probe. This probe format incorporates an internal Nova quencher modification between bases 9 and 10 of the probe sequence in addition to the 3’ terminal BHQ modification. As a result, these probes are very efficiently quenched even in longer probe sequences, allowing greater flexibility of design without sacrificing performance.
   • Is the melting temperature ( T_M) of the probe near the recommended 68 °C? If the probe T_M is too low, then the probe will not hybridize well resulting in poor signal generation.

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

Why is my BHQ®-Pulsar® 650 probe not detected in a singleplex reaction on my LightCycler?

Why do I get different Ct values for the same probe sequence when labeled with a different dye?

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.

I am a beginner at real-time qPCR. Does LGC Biosearch Technologies have information which will help me to design my assay?

For an overview of available Dual-labeled BHQ® probe types, their mode of action and basic design guidelines, you may download our Fluorogenic Probes and Primers Brochure.

For an in depth discussion on qPCR, including probe and primer design, we recommend reading the book entitled 'A-Z of Quantitative PCR' edited by Stephen A. Bustin.

Additional resources are available on-line, including the website 'REAL-TIME PCR' maintained by M. Tevfik Dorak, MD, Ph.D., which offers a review of major topics for qPCR and historical links to valued information.

For MIQE guidelines on experiment design, please see the original publication entitled, "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments", by Bustin et al.

For applications and the most recent methods in gene expression analysis, visit the website named 'www.Gene-Quantification.info: The Reference in qPCR - Academic & Industrial Information Platform'. This site offers many links to additional resources on qPCR.

Does LGC Biosearch Technologies have software available for designing probes and primers?

For the design of qPCR primers and probe sets, we offer our RealTimeDesign™ (RTD™) software. The RTD software is free, easy to use and can be accessed directly through your web browser. Our software offers a choice of design modes: an 'Express Mode' with pre-set parameters and a 'Custom Mode' in which the parameters can be adjusted by the user. There is an additional 'Batch Mode' which facilitates the design of up to 10 assays in series. 

Is there a formula for calculating the efficiency of a qPCR reaction?

For a singleplex reaction, the efficiency of qPCR is calculated as follows:

Efficiency = 10^(-1/slope) - 1

The slope is derived from a graph of Cycles to Threshold (Ct) values plotted against the Log₁₀ of the template amount. A slope of -3.32 indicates an amplification efficiency of 100%.

Resource: Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. David G. Ginzinger. Experimental Hematology 30 (2002): 503-512

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.

How do I determine which design mode to use in LGC Biosearch Technologies' RealTimeDesignâ„¢ software?

The RealTimeDesign™ (RTD™) software offers three different levels of user control to accommodate a range of needs:

Express Mode - is designed with simplicity in mind. This mode does not require any input from the user other than sequence submission and label selection.

Custom Mode - is designed for the advanced user who wishes to inspect, include or exclude certain oligonucleotide candidates from each stage of the design process. This mode provides the user with access to the parameter settings for advanced control.

Batch Mode - is similar to Express Mode however it allows users to initiate designs against as many as 10 target sequences and close the browser while they wait. An email will be sent alerting the user that the designs have completed.

All designs may be reviewed on the Design Run History page.

How do I enter in a SNP, MNP or InDel sequence into the RealTimeDesignâ„¢ software?

Within the RealTimeDesignâ„¢ software, how do I find the primers and probe sequences after I design them?

I have my own primer designs. Can LGC Biosearch Technologies' RealTimeDesignâ„¢ (RTDâ„¢) software design only the probe for me?

To design a probe for compatibility with pre-designed primers, select application and design mode, then select the 'Include/Exclude' box at the bottom of the second pull-down menu. In the next screen, users may input the desired primer sequences into the 'Anchored' column of the oligonucleotide table. The RealTimeDesign™ (RTD™) software will then proceed to design a Dual-labeled BHQ® or BHQplus® probe within those predefined primer sequences.

Note: LGC Biosearch Technologies does not recommend using primers designed outside of the RTD software because primers used for other applications (e.g. gel electrophoresis) are often inappropriate for qPCR. The RTD software uses parameter settings that are proven to design primers and probe sets with optimal performance.

Is there any way of designating a specific region of my sequence for the RealTimeDesignâ„¢ software to design my primers, probe and assay?

What is meant by 'Tandem Repeats' and 'Mask Tandems' when using the RealTimeDesignâ„¢ software?

What does the 'Overall Rank' score in your RealTimeDesignâ„¢ software represent?

RealTimeDesign™ presents all candidate primers, probes, and assays in order of descending rank score. The rank score represents how closely the design matches the ideal values for each parameter setting. This aggregate value is principally used by the software to advance those candidate oligos that are most optimal, to ultimately present a single assay that is the most highly ranked.

What is the 'Failure Count Data' in your RealTimeDesignâ„¢ software?

I am a beginner at real-time qPCR. Does LGC Biosearch Technologies have information which will help me to design my assay?

For an overview of available Dual-labeled BHQ® probe types, their mode of action and basic design guidelines, you may download our Fluorogenic Probes and Primers Brochure.

For an in depth discussion on qPCR, including probe and primer design, we recommend reading the book entitled 'A-Z of Quantitative PCR' edited by Stephen A. Bustin.

Additional resources are available on-line, including the website 'REAL-TIME PCR' maintained by M. Tevfik Dorak, MD, Ph.D., which offers a review of major topics for qPCR and historical links to valued information.

For MIQE guidelines on experiment design, please see the original publication entitled, "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments", by Bustin et al.

For applications and the most recent methods in gene expression analysis, visit the website named 'www.Gene-Quantification.info: The Reference in qPCR - Academic & Industrial Information Platform'. This site offers many links to additional resources on qPCR.

Does LGC Biosearch Technologies have software available for designing probes and primers?

For the design of qPCR primers and probe sets, we offer our RealTimeDesign™ (RTD™) software. The RTD software is free, easy to use and can be accessed directly through your web browser. Our software offers a choice of design modes: an 'Express Mode' with pre-set parameters and a 'Custom Mode' in which the parameters can be adjusted by the user. There is an additional 'Batch Mode' which facilitates the design of up to 10 assays in series. 

What are the differences in parameter settings between the various modes within the RealTimeDesignâ„¢ software?

How is the Tm calculated in LGC Biosearch’s Technologies' RealTimeDesign™ software?

Our RealTimeDesign™ (RTD™) uses the SantaLucia "unified" nearest neighbor thermodynamic parameters in the algorithm to calculate melting temperature (or T_M). There are often discrepancies between the T_M values predicted using RTD and those of other programs due to different thermodynamic values and also different concentrations for the assay components. These differences are further explained in the following reference: Comparison of different melting temperature calculation methods for short DNA sequences. Alejandro Panjkovich and Francisco Melo. Bioinformatics 2005 21(6):711-722; 2004 doi: 10.1093/bioinformatics/bti066

For more information, consider the publication: "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics", by John SantaLucia, Jr.

We also recommend a general web tool to model oligo binding called DINAMelt, which can be adapted to a variety of experimental conditions. DINAMelt draws upon the same SantaLucia thermodynamic values as RTD, and a description of its algorithms may be found in the following publication: DINAMelt web server for nucleic acid melting prediction. Markham NR, Zuker M. Nucleic Acids Res. 2005 Jul 1;33:W577-81.

How do I order the primers and probe I have designed using the RealTimeDesignâ„¢ software?

What are the recommended storage conditions for Dual-labeled BHQ® probes?

How do freeze thaw cycles affect oligonucleotides?

I left my oligonucleotides sitting on the lab bench over the weekend. Are they still ok to use?

What is your recommended method for reconstituting and storing oligonucleotides?

To reconstitute dry oligonucleotides:

To prepare a 100 µM stock of reconstituted probe: Multiply the total nmol value provided on the Certificate of Analysis, by 10. The resulting number will be the volume of diluent (in microliters) to add to your probe. Once resuspended, the probe will be 100 µM and represents a stock solution.

For example: If the delivered amount was 20 nmol, then:
(20) x (10) = 200 microliters is needed to reconstitute the oligo to 100 µM.

1. We recommend a short centrifugation of the product tube to ensure the oligonucleotides pellet is at the bottom.
2. Resuspend the product in an appropriate volume of solution such as TE buffer (10 mM Tris, 1mM EDTA, pH 8), to achieve a stock concentration of 10 µM or more, ideally 100 µM.
3. Allow the oligo to rehydrate for several minutes at room temperature and vortex for 15 seconds.
4. Product should be exposed to a minimum of freeze/thaw cycles to avoid oligo degradation. We recommend aliquoting the stock solution into multiple aliquots for storage at -20 °C or cooler, in the dark. When stored in this manner, we anticipate product stability for one year from delivery.

Note: Dual-labeled BHQ® probes are sensitive to photobleaching so protect from light exposure.l.

What buffer should I resuspend my oligonucleotide in?

For oligonucleotide suspension, we recommend preparing stock and working solutions using a TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) made with nuclease-free water. The EDTA serves to protect against microbial contamination. If your experiment cannot tolerate EDTA, you may use 10 mM Tris-Cl buffer. Suspension in water alone should be limited to nuclease-free water at physiological pHs, but is not recommended. Acidic conditions can lead to degradation of the oligonucleotide through depurination.

Notes: Fluorophores are sensitive to photobleaching. To minimize their exposure to light, we recommend using amber microtubes, wrapping the tube in foil, or else placing clear tubes into a box which is impermeable to light. Avoid freeze/thaw cycles by making working solutions and storing them in aliquots at -15 °C or cooler.

How do I determine the volume of buffer to add to my probe to prepare a stock concentration?

I received my oligonucleotide order, but the vial looks empty. Where is the oligonucelotide?

Why were my oligonucleotides shipped at room temperature?

How should orders be placed for shipment to locations outside the United States?

LGC Biosearch Technologies maintains distribution agreements with partners in countries listed on our Distributors webpage. For all other countries, LGC Biosearch will ship internationally subject to local customs restrictions. All payments must be in US dollars.

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

Where can I find usage information and chemical properties for Biosearch Technologies' products?

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.

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 are fluorescent labels on Dual-labeled BHQ® probes accounted for when measuring the absorbance?

Extinction coefficients are a prediction of each oligo’s molar absorbance and the standard method to calculate concentration. To estimate the extinction coefficient of modified oligonucleotides, we use the following equation: ε260 = [(Sum of ε260 for all bases) + (ε260 for all modifications)] x 0.9, to adjust for hyperchromicity. Individual extinction coefficients for each modification are available under the Technical Specs tabs on our Oligo Modifications webpages. The extinction coefficient found on the Certificate of Analysis for a custom oligo includes all dye and other modifications in the presented value. Simply use this value and measure the OD at 260 nm to calculate oligo concentration according to Beer’s Law. 

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 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 order a Stellaris® probe set that is not available for online ordering?

We have a Stellaris excel order form available in case you already have probe sequences on file.

Stellaris Probes Excel Form

Submit a completed form to our customer service team at info@biosearchtech.com.

Please also contact info@biosearchtech.com if you wish to inquire about the following:

• Labeling your Stellaris probe set with a modification not advertised on the website
• Ordering a DesignReady probe set with an unlisted dye

For more product information about Stellaris probe sets, please visit: https://www.biosearchtech.com/products/rna-fish

Can LGC Biosearch Technologies' probes (and dyes) be used for fluorescence in situ hybridization (FISH)?

LGC Biosearch Technologies offers Stellaris® RNA FISH probes and associated products for RNA FISH. We do not currently support DNA FISH applications. Historically, FISH probes were cDNA labeled enzymatically with multiple fluorophores. A single synthetic oligonucleotide labeled with a fluorophore is not sufficiently bright for reliable visualization under a microscope and therefore multiple probes are necessary. With Stellaris RNA FISH Probes, multiple probes hybridize in series along the transcript of interest. The combined fluorescence from the probe set hybridized to the transcript can be seen as a diffraction-limited spot in a widefield fluorescence microscope, allowing both the location of the RNA and the copy number per cell to be revealed.

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.

Can Stellaris® RNA FISH Probes detect DNA targets?

Stellaris RNA FISH Probes hybridize to RNA. Genomic (double stranded) DNA remains inaccessible as no denaturation step is performed to unwind the DNA. Further, the Tm of the short, single-stranded probes is lower in DNA than in RNA and would therefore be competed away by the complementary strand. Simultaneous RNA and DNA detection using different probe types is not currently supported because the denaturation step needed to unwind DNA will remove the Stellaris RNA FISH probes.

Which fluorescent dye should I choose for my Stellaris® RNA FISH Probe set?

Selection of the fluorescent dye for your Stellaris RNA FISH Probe set should be chosen to best match the specifications of the available band-pass filters sets on the microscope you will be using. Visit our Stellaris Dyes and Modifications page for specific recommendations and considerations for each of our dyes. To learn more about how to align the dye spectra with your filters, check out our blog here (Imaging Stellaris Assays: Get to Know Your Microscope). It is equally important to consider the autofluorescence of the sample types you plan to study. Autofluorescence in tissue samples is more pronounced in the green wavelengths. Fluorescein, as an example, absorbs around 450 nm and emits around 520 nm.. It is easier to discern true signal from autofluorescence if longer wavelength fluorophores, such as Quasar® 570, 670, or CAL Fluor® Red 610 dyes, which emit in the red or far-red, are used instead. Additionally, be mindful that if you plan on using a transgenic cell line that may be expressing a fluorescent protein like GFP or tdTomato, you want to choose a fluorophore with a different fluorescence maximum. We recommend that you use Chroma’s online dye and filter selection software to help you choose.

Can I use a confocal microscope with Stellaris® RNA FISH probe sets?

We recommend the use of wide-field fluorescence microscopes as an entry point for researchers new to Stellaris and microscopy. However, more advanced microscopists have successfully employed confocal microscopy for imaging their Stellaris RNA FISH assays. Confocal microscopy uses point illumination to limit the focal plane for imaging. While this technique restricts light that is out of focus, it also diminishes the sensitivity of low-light level imaging.

To best detect individual transcript molecules, we encourage the use of conventional wide-field fluorescence microscopes with a 60-100x, 1.3 NA or greater, oil-immersion objective and a cooled CCD camera. The light source should be a mercury or metal-halide lamp (e.g., ExFo Excite, Prior Lumen 200). We recommend starting with a 1 second exposure time. Check out our blog article for tips on imaging Stellaris RNA FISH assays.

Can Stellaris® RNA FISH probes be used for multiplexing?

Stellaris RNA FISH Probes may be used to detect several targets simultaneously.  The number of possible targets that can be detected depends on the available microscope filters that can spectrally separate each dye.  Dyes with minimal spectral overlap can be combined as long as they are carefully matched to those filter sets! We recommend verifying the performance of each probe set individually before starting your multiplex experiments. Visit our Stellaris Dyes and Modifications page for specific dye multiplexing recommendations.

Can I design a Stellaris® FISH RNA probe set to detect small RNAs?

The Stellaris Probe Designer software, available on our website, outputs a probe set with up to 48 oligonucleotides which are each 18-22 nucleotides long. A target of at least 1 kb in length is needed for the design of a full 48-oligonucleotide probe set. By reducing the number of probes in a set, the Stellaris technology can be used with target sequences as short as 600 nucleotides. If your sequence is significantly shorter, then the Stellaris method is less likely to provide for reliable single molecule detection. A shorter sequence may also be suitable if your target is found in clusters, such as at the site of transcription, or in Cajal Bodies. No matter what your design challenge might be, we want to help further your research with Stellaris, so please reach out to our design team at techsupport@biosearchtech.com to learn more about whether your target of interest can be detected by Stellaris RNA FISH.

How long does it take to manufacture and ship Stellaris® RNA FISH Probe sets?

Turnaround time is 5 to 7 business days for manufacture, with one additional business day for delivery via FedEx Priority. Stellaris RNA FISH probe sets are shipped dry so neither dry ice nor cold packs are required.

LGC Biosearch Technologies’ Quasar® 670 dye is listed as an alternative to the Cyâ„¢ 5 dye. Is it more resistant to photobleaching?

No, both Quasar® 670 and Cy5 share similar photobleaching characteristics. Like the original Cy5 dye, our Quasar 670 is not sulfonated and is based on the same indocarbocyanine as the Cy5 dye giving it nearly identical characteristics. We recommend commercial mounting media such as Vectashield® and imaging longer wavelength dyes first to help preserve the fluorescence signal. Visit our Stellaris Dyes and Modifications page for more information.

What happens to the yield for each Stellaris® oligonucleotide if my submitted sequence is not long enough to return 48 probe sequences?

The final delivered amount of each oligonucleotide in a Stellaris® RNA FISH Probe set will vary according to the total number of oligonucleotides designed. If there are less than 48 probes in the set, then the total amount for each will increase such that the final blended amount is 5 nmol. Additionally, it is not usually necessary to have 48 distinct probes to distinguish an RNA target and visualization of single RNA molecules is possible with less than 48 probes in a set.

How do I know that the Stellaris® RNA FISH Probe set is binding specifically to RNA and not DNA?

When setting up my Stellaris® RNA FISH assay, why do I have to adhere my cells to a no. 1 coverslip?

For optimum clarity of signal from the individual transcripts, the sample needs to be as close as possible to the lens, and located in a neat uniform plane. The distance to detection includes the thickness of the glass to which the cells are adhered. When using a thicker glass surface, such as a no. 1.5 coverslip, the sensitivity is reduced and distinct Stellaris RNA FISH signals are more difficult to detect.

Can I use commercial antifade media with the Stellaris® method?

Our dyes are compatible with many commercial mounting media such as Vectashield® Soft and Hard set, Prolong® Gold, Prolong® Diamond, and Slow Fade® Diamond.  We recommend using Vectashield Mounting Medium (Vector Labs, catalog #H1000). Be aware that some commercial mounting media require the sample to cure for an extended period of time. Though this may be convenient for your experimental workflow,we recommend imaging your sample immediately after mounting, as fluorescence will naturally degrade over time. Visit our Stellaris Dyes and Modifications page for more information.

Can I use Stellaris® RNA FISH Probes to distinguish RNAs from genes with high homology?

Studies by LGC Biosearch Technologies and our collaborators have shown that RNAs from genes with high homology can be distinguished by Stellaris RNA FISH. Most gene homology resides in the coding region, and if the untranslated regions can be used as targets, then targets with high overall homology can be more easily discriminated. Under the conditions of hybridization in the Stellaris RNA FISH method, oligonucleotides containing two or more mismatches to the target sequence will hybridize only weakly. To identify and eliminate cross-reacting sequences, we recommend applying bioinformatic approaches like BLAST or a multiple sequence alignment.

Can the Stellaris® method be used for whole mount samples?

The Stellaris method has been successfully employed to examine RNAs in whole-mount zebrafish embryo, C. elegans, and Drosophila as well as in cultured cells, frozen tissue and FFPE (Formalin-fixed Paraffin-embedded) tissue slices 4-20 micrometers in thickness. Visit our Stellaris Citation Center to see all publications citing Stellaris RNA FISH in whole mount applications. Below, you can find examples of publications using Stellaris RNA FISH probes in whole mount samples.  If a whole mount sample is of appropriate thickness, then there is potential for the Stellaris RNA FISH technology to perform well. The Stellaris RNA FISH Probes consist of oligonucleotides on average 20 nucleotides in length, which penetrate tissue better than long DNA probes, or assemblies for indirect detection. In thicker tissue sections, i.e. > 20 µm, Stellaris RNA FISH Probes may offer qualitative results sans the ability to effectively count transcripts. Stellaris RNA FISH Probes may not always afford a sufficient signal to visualize transcripts in thick sections.

Oka Y, Sato TN. Whole-mount single molecule FISH method for zebrafish embryo. Scientific Reports. 2015;5:8571. doi:10.1038/srep08571.

Ji N, van Oudenaarden A. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos. Wormbook. 2012;13:1-16 doi: 10.1895/wormbook.1.153.1

Xu H, Sepúlveda LA, Figard L, Sokac AM, Golding I. Combining protein and mRNA quantification to decipher transcriptional regulation. Nature Methods. 2015;12(8):739-42. Doi10.1038/nmeth.3446

Do you offer a trial Stellaris® RNA FISH probe set for me to test out the method in my laboratory?

LGC Biosearch Technologies offers Stellaris ShipReady probe sets, with 1 nmol of product delivered, which can be used to establish and troubleshoot the Stellaris method in your laboratory. These ShipReady positive controls provide an inexpensive way to demo the method before committing to the synthesis of a custom probe set. The Stellaris RNA FISH protocols we present on the website are optimized for most systems and we recommend adherence to them. Subtle omissions can have a profound impact on product performance.

What is the consequence (and cost) if I order a Stellaris® RNA FISH probe set with fewer than 48 oligos?

In general, the Stellaris RNA FISH method produces better results when using probe counts over 25. By default, the software proposes up to 48 probes when the transcript is sufficiently long. If ordering fewer than 48 probes, the quantity of each oligonucleotide in a Stellaris RNA FISH Probe set will vary to maintain a final blended amount of 5 nmol. The cost of a Stellaris RNA FISH Probe set remains the same regardless of the number of distinct sequences in the probe set.

Are Stellaris® RNA FISH Probes comprised of DNA, RNA, or non-standard nucleotides?

Stellaris® RNA FISH probes are comprised of DNA oligonucleotides, each singly-labeled with a fluorophore. Their simple structure and small size allow for sample permeabilization without protease treatment. As a result, the Stellaris method is less cumbersome than other FISH methods and more easily integrates with complementary detection techniques, such as immunofluorescence.

Can the Stellaris® RNA FISH method be combined with immunofluorescence?

Stellaris RNA FISH can be combined with immunofluorescence (IF), although some antibodies may not be compatible with FISH conditions. The fixation method used will affect the integrity of cellular structures and macromolecular assemblies of RNA and protein. It may also affect the accessibility of the epitope to be interrogated. Three approaches have been successful in combining Stellaris RNA FISH with IF: 1) Just adding in the IF-antibody/antibodies into the hybridization and/or wash buffers. 2) Performing the IF, then re-fixing the sample with formaldehyde, and then performing the RNA FISH. 3) Performing RNA FISH, then re-fixing the sample, and then performing the IF.

The following methods article and supplement outlines a protocol to combine the Stellaris RNA FISH method with immunofluorescence in cells, with details on page 4 of the Supplemental Material:

Imaging Individual mRNA Molecules Using Multiple Singly Labeled Probes. (2008) Raj, A.; van den Bogaard, P.; Rifkin, S.A.; van Oudenaarden, A.; Tyagi, S. Nature Methods 5(10), 877-9.

Another excellent paper combining IF with Stellaris RNA FISH is:

Visualization of Single mRNAs Reveals Temporal Association of Proteins with microRNA Regulated mRNA. (2011) Shih, J.D.; Waks, Z.; Kedersha, N.; Silver, P.A. Nucleic Acids Research 39(17), 7740-9.

Why is autofluorescence obscuring my Stellaris® RNA FISH signal?

Most cells and tissue types have natural autofluorescence that is more pronounced in the green region of the visible spectrum. It is easier to discern true signal from autofluorescence if longer wavelength fluorophores such as Quasar® 570/670 or CAL Fluor® Red 610 are used.  The signal from shorter wavelength dyes, such as fluorescein (emission near 520 nm), is more difficult to detect due to the autofluorescence.

Autofluorescence is generally more evident in tissue samples, is more pronounced in certain cultured cell types, and can have very broad emission that spans the visible spectrum. For example, fluorescence from lipofuscin bodies can be detected from 360 nm to 650 nm.  Fluorescence from the lipofuscin bodies is mostly seen as cytoplasmic perinuclear spots which can be distinguished from true single molecules of RNA by being slightly larger, brighter, and by having fluorescence over a broad range of the spectrum. When you’re searching for your RNA of interest in tissue, it is especially important to distinguish the Stellaris RNA FISH signals from autofluorescent features. One way to accomplish this is to collect the light in a secondary unused filter. This will let you confirm that your RNA of interest is only present when you specifically excite the fluorophore you labeled it with.  For example, if your probe is labeled with Quasar 670 that means you will excite this dye near 647 nm and it will emit fluorescence at 670 nm.  If you try to excite that same molecule at a shorter wavelength with an unused filter, say FITC near 470 nm, you should not see the fluorescence in the same location.  If you do see fluorescence using multiple filters or with filters which are incorrect for your dye, then you are likely seeing autofluorescence and not a true RNA molecule.  For more information about the prevalence and origin of autofluorescence, please visit the Nikon Microscopy U website.

In addition, the degree of autofluorescence will depend on the fixation method used. Formaldehyde tends to crosslink fluorescent enzyme co-factors, such as flavins, whereas fixation with methanol/acetic acid tends to release them and wash them away from the sample. Prolonged fixation at elevated temperatures tends to exacerbate the autofluorescence. To assess the overall background fluorescence and to determine the contribution of non-specific probe binding, we recommend imaging a second sample in parallel that has not undergone probe hybridization - a no probe control.

What types of controls do you recommend for Stellaris® RNA FISH assays?

The type of Stellaris® RNA FISH controls needed is dictated by the experiment set up and the preferences of the researcher. Whereas sense or scrambled sequences are useful in antisense or RNAi silencing, they do not serve as an appropriate negative control in Stellaris RNA FISH. An ideal negative control would be a specific probe set against an RNA that normally is not expressed in the cell/tissue sample to be tested. Alternatively, you may consider using a probe set targeting the RNA from a gene from a different organism and which is absent from your sample, e.g. GFP. Please read this article on how controls can demystify your Stellaris RNA FISH experiment for more details.

Formalin-fixed paraffin embedded (FFPE) tissue may suffer from RNA degradation, sometimes very significantly. In such cases, detection of an RNA from a reference gene can be useful for verification of the experimental technique. One commonly used reference gene is glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We make available several Stellaris ShipReady probe sets that can serve as controls, including human and mouse GAPDH, and DesignReady probe sets for many other organisms.

What is the purpose of the different masking levels available in the Stellaris® Probe Designer? Which level should I use?

The Stellaris Probe Designer software lets you design probe sets with adjustable specificity. Please read this article on designing Stellaris RNA FISH probes for step by step instructions on increasing probe count. At levels 0, 1, and 2 the target sequence remains non-masked and oligos are laid down according to the optimal T_M and other parameters selected (number of oligos, length, and spacing). At levels 1 and 2, oligos with known difficult sequences or simple repeats such as G-strings, are not allowed. At levels 3 and up, species-specific masking is performed to avoid cross-hybridization to RNAs commonly expressed at high to very high levels.

We advise selecting the most stringent setting possible for your organism, while still generating a full probe count of 24 or more. In the case that you are not able to generate at least 24 probes in your set, you should try decreasing the masking level and trying again. If your species is not listed in the pull down menu, then level 2 is the most stringent available for "other" organisms. The software will not anticipate every scenario and additional bioinformatic analysis is prudent, according to the particulars of the investigation. We advise both careful choice of the target RNA, and BLASTing individual probe sequences against the appropriate database, especially if the intent is to generate a probe set that tolerates or discriminates between similar transcripts.