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Freedom™ Dyes
With Freedom Dyes, you have license-free access to patented fluorescent dyes. We offer fluorophores spanning the commonly used excitation and emission wavelengths. These dyes can be paired with IDT's proprietary single or dual dark quenchers (ZEN™ and TAO™).
Product Details
Freedom fluorescein dyes
| Dye | 5' | Internal | 3' | Excitation (nm) | Emission (nm) | Alternative |
|---|---|---|---|---|---|---|
| 6-FAM (Fluorescein) | details | – | details | 495 | 520 | N/A |
| 6-FAM (NHS Ester) | details | – | details | 496 | 516 | N/A |
| Fluorescein dT | details | details | details | 495 | 520 | N/A |
| HEX | details | – | – | 538 | 555 | Yakima Yellow® |
| Yakima Yellow | details | - | - | 524 | 551 | HEX |
| JOE™ (NHS Ester) | details | – | details | 529 | 555 | MAX (NHS Ester) |
| MAX | details | – | details | 524 | 557 | JOE (NHS Ester) |
| TET | details | – | – | 522 | 539 | N/A |
Yakima Yellow is registered trademark of ELITech Group; JOE is a trademark of Thermo Fisher Scientific.
6-FAM (Fluorescein): A single isomer derivative of fluorescein. FAM is a commonly used fluorescent dye attachment for oligonucleotides and is compatible with a variety of fluorescence detection equipment. It becomes protonated and has decreased fluorescence below pH 7; it is typically used in the pH range 7.5–8.5. FAM can be attached to 5' or 3' end of oligos.
6-FAM (NHS Ester): A post-deprotection reaction is used to attach this fluorophore to the oligonucleotide. This is commonly used to modify RNA oligos with FAM.
Fluorescein dT: Fluorescein is attached to position 5 of the thymine ring by a 6-carbon spacer arm.
SUN: A VIC® (ThermoFisher Scientific) equivalent (emission maximum of 554 nm) that is recommended for use in multiplexed assays when added to the 5' end of a dual-labeled 5' nuclease probe for qPCR.
HEX: Hexachlorofluorescein is a chemical relative of fluorescein that is commonly used for multiplexed assays with FAM and TET. HEX can be added only to the 5' end of an oligonucleotide.
JOE (NHS Ester): JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein) is available only as an NHS ester and must be attached to an amino-modified oligonucleotide. JOE is frequently used in multiplex reactions with FAM.
MAX (NHS Ester): Most often used to multiplex with FAM. It can be used as a VIC substitute because its spectral profile is nearly identical to VIC. Importantly, MAX is efficiently excited by either a 488 nm laser or a broad emission light source; therefore, it can be used with most commercial detection platforms.
TET: Tetrachlorofluorescein is a chemical relative of fluorescein that is commonly used for multiplexed assays with FAM and HEX. TET can be added only to the 5' end of an oligonucleotide.
Freedom Cy dyes
| Dye | 5' | Internal | 3' | Excitation (nm) | Emission (nm) | Alternative |
|---|---|---|---|---|---|---|
| Cy 3 | details | details | details | 550 | 564 | TYE™ 563 |
| Cy 5 | details | details | details | 648 | 668 | TYE 665 |
| Cy 5.5 | details | – | details | 685 | 706 | TYE 705 |
Cy 3: A standard orange-fluorescent label for nucleic acids used in imaging, flow cytometry, and genomic applications.
Cy 5: A standard far red-fluorescent label for nucleic acids used in imaging, flow cytometry, and genomic applications.
Cy 5.5: Used as a fluorescent label in 5' nuclease probes for qPCR, Scorpion® primers (Premier Biosoft), and Molecular Beacons. Cy 5.5 can also be used to label DNA oligos for use as hybridization probes in other applications, such as fluorescent in situ hybridization (FISH).
Freedom rhodamine dyes
| Dye | 5' | Internal | 3' | Excitation (nm) | Emission (nm) | Alternative |
|---|---|---|---|---|---|---|
| ROX | details | – | details | 588 | 608 | ATTO Rh101 (NHS Ester) |
| TAMRA | – | – | details | 559 | 583 | N/A |
| TAMRA (NHS Ester) | details | details | details | 559 | 583 | N/A |
| TEX™ 615 | details | – | details | 596 | 613 | Texas Red®-X (NHS Ester) |
TEX 615 is a trademark of Thermo Fisher Scientific, and Texas Red is a registered trademark of Molecular Probes, Inc.
ROX (NHS Ester): ROX (carboxy-X-rhodamine) is available only as an NHS ester; therefore, it must be attached to an amino-modified oligonucleotide. It has spectral properties similar to Texas Red.
TAMRA: This phosphoramidite modification is only available at the 3' end of an oligonucleotide. This was used as a quencher in first generation real-time PCR.
TAMRA (NHS Ester): The internal and 3' versions of this modification are attached to the oligonucleotide through a dT base; therefore, a dT nucleotide will be added at the position of these modifications. To avoid adding an extra nucleotide, replace an existing T nucleotide in your sequence with the required modification.
TEX 615: A red wavelength dye commonly used in multiplex reactions with FAM or in fluorescence-activated cell sorting (FACS) and microscopy applications using the rhodamine filter. TEX 615 can be used as a direct substitute for Texas Red or CAL Fluor 610.
Freedom ATTO™ dyes
| Dye | 5' | Internal | 3' | Excitation (nm) | Emission (nm) | Alternative |
|---|---|---|---|---|---|---|
| ATTO 488 | details | – | details | 502 | 522 | Alexa Fluor® 488 |
| ATTO 532 | details | – | details | 534 | 554 | VIC® |
| ATTO 550 | details | – | details | 560 | 575 | Alexa Fluor 555 |
| ATTO 565 | details | – | details | 570 | 591 | PET™ |
| ATTO Rho101 | details | – | details | 592 | 609 | ROX |
| ATTO 590 | details | – | details | 602 | 624 | Alexa Fluor 594 |
| ATTO 633 | details | – | details | 635 | 653 | LIZ® |
| ATTO 647N | details | – | details | 649 | 662 | Alexa Fluor 647 |
Alexa Fluor fluorophores are registered trademarks of Molecular Probes, Inc.; VIC and LIZ are registered trademarks of Thermo Fisher Scientific; PET is a trademark of Thermo Fisher Scientific.
ATTO dyes are patented fluorescent markers that feature strong absorption, high photostability, and high fluorescence quantum yield [1]. ATTO dyes can be used as substitutes for dyes such as ROX, VIC, LIZ, and Alexa Fluor dyes. Common applications for ATTO dyes include fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET), and dual-labeled probes used in real-time PCR experiments. For real-time PCR applications, dye calibration will be needed for each real-time PCR instrument.
Other Freedom Dyes
| Dye | 5' | Internal | 3' | Excitation (nm) | Emission (nm) | Alternative |
|---|---|---|---|---|---|---|
| TYE 563 | details | – | details | 549 | 563 | Cy 3 |
| TYE 665 | details | – | details | 645 | 665 | Cy 5 |
| TYE 705 | details | – | – | 686 | 704 | Cy 5.5 |
TYE fluorophores are licensed from Thermo Fisher Scientific.
TYE dyes are proprietary fluorophores from IDT that are available for commonly used dye wavelengths. The numerical value in the name of these dyes represents the emission wavelength when attached to an oligo.
TYE 563: A bright fluorescent dye that can be used in microarray applications. TYE 563 can be used as a direct substitute for Cy 3.
TYE 665: A bright fluorescent dye that can be used in microarray applications. TYE 665 can be used as a direct substitute for Cy 5.
TYE 705: A bright fluorescent dye that can be used in microarray applications. TYE 705 can be used as a direct substitute for Cy 5.5.
Additional fluorophores (which may include licensing restrictions) are available. Click here to see our full catalog of fluorophore modifications.
Resources
Frequently asked Questions
Will polyacrylamide gel electrophoresis damage a Cy-labeled oligonucleotide?
We do not recommend PAGE purification of Cy-labeled oligonucleotides. Ammonium persulfate, a cross linking agent used in place of urea, is a component of the acrylamide gel that can damage the dye. Ammonium persulfate oxidizes the double bonds in the chain between the rings, affecting the dye's ability to flouresce when excited. HPLC purification, however, does not oxidize Cy dyes and is required for these modifications.
Why is PAGE purification not available with fluorescent dyes?
We do not recommend PAGE purification for dye labeled oligos due to the presence of ammonium persulfate, a component of the PAGE gel. Ammonium persulfate can break linkages and may damage the ring structures that comprise many fluorescent dyes. At IDT, PAGE gels are exposed to UV light when the oligos are excised from the gel. UV light exposure can also damage fluorescent dyes. HPLC purification provides a pure product without damaging fluorescent modifications.
Which dyes are compatible with my thermal cycler?
To determine what dyes are compatible with your instrument, download our Real-time qPCR assay design guide list.
Which dyes are available for use with the IDT ZEN quencher?
Currently, the IDT ZEN™ quencher is available with FAM, HEX, TET, Yakima Yellow® (ELITech Group), JOE™ (ThermoFisher), MAX™, and SUN™ fluorophores. Double-quenched probes have significantly lower background fluorescence, especially for longer probes.
The ZEN quencher is available on PrimeTime™ qPCR Probes and on the probes supplied with PrimeTime qPCR Probe Assays. For any special oligonucleotide requests that include the ZEN quencher but are not found on our website (non-catalog requests), please contact applicationsupport@idtdna.com.
For more information, see Double-quenched probes increase qPCR sensitivity and precision.
ZEN, SUN, MAX, and PrimeTime are trademarks of IDT.
Which dye label provides the brightest signal and where in the oligo should it be positioned?
The dyes with the most intense signals that IDT offers are the Alexa Fluor and ATTO dye classes of fluorescent dyes. Signal intensity is not effected by position within the oligo sequence. Green, yellow, and some orange dyes can be partially quenched by guanine bases. Therefore, if you decide to order a dye in that range, you should try not to position the dye immediately next to a guanine base. Read more details about this quenching effect.
Where on a nucleotide are Cy dyes attached?
5′ and 3' Cy dyes are attached to the hydroxyl group of the ribose via a phosphodiester bond.
Internal Cy dyes are attached to the backbone via the phosphodiester bonds of the bases between which the dyes occur.
Internal Cy dyes are attached to the backbone via the phosphodiester bonds of the bases between which the dyes occur.
What modification can I use in place of VIC®?
VIC® is a proprietary modification owned by Applied Biosystsems (Life Technologies). SUN™ is a fluorophore offered by IDT that is a molecular equivalent to VIC and available for qPCR probes. For other applications, fluorophores such as HEX, MAX™ (IDT), or Yakima Yellow® (ELITech Group) are available license-free alternatives.
However, be sure that your fluorescent detection instrument is calibrated for the dye you choose or can detect the dye once calibrated. If choosing dye combinations for multiplex qPCR, IDT offers the Multiplex qPCR Dye selection tool will help you find the best combination of dyes for your specific qPCR instrument.
However, be sure that your fluorescent detection instrument is calibrated for the dye you choose or can detect the dye once calibrated. If choosing dye combinations for multiplex qPCR, IDT offers the Multiplex qPCR Dye selection tool will help you find the best combination of dyes for your specific qPCR instrument.
What is the stability of fluorescently labeled oligos and their fluorescent modifications?
The stability of fluorescently labeled oligos and fluorophore function is similar to the stability of a standard oligo.
For optimal stability, oligos that are to be stored long term should be stored frozen, at -20°C. If the oligos are going to be resuspended, storage in a TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5–8.0; such as IDTE) is better than using water (non-DEPC treated). The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures (see the Technical Report, Oligonucleotide Stability Study, for the data).
With fluorophores, it’s important to remember that exposure to natural light or ozone can negatively impact stability. If fluorescently modified oligos will be exposed to sustained light, protective cover from the lighting source (e.g., using brown tubes and/or foil covering) should be considered.
For optimal stability, oligos that are to be stored long term should be stored frozen, at -20°C. If the oligos are going to be resuspended, storage in a TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5–8.0; such as IDTE) is better than using water (non-DEPC treated). The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures (see the Technical Report, Oligonucleotide Stability Study, for the data).
With fluorophores, it’s important to remember that exposure to natural light or ozone can negatively impact stability. If fluorescently modified oligos will be exposed to sustained light, protective cover from the lighting source (e.g., using brown tubes and/or foil covering) should be considered.
What dyes should I select for my qPCR assay?
IDT offers a free Real-time qPCR assay design guide that contains additional information on available dyes, and instrument compatibility.
On your website, I see that there are two different extinction coefficients for various dyes. Which one should I use?
The extinction coefficient "at maximum absorbance" refers to the extinction value that a given molecule has at its unique absorption maximum.
The extinction coefficient "not at absorbance maximum" refers to the extinction coefficient at 260 nm, which is the wavelength used to determine for optical density quantitation.
See the DECODED article about quantifying your oligo at: https://www.idtdna.com/pages/education/decoded/article/oligo-quantification-getting-it-right
The extinction coefficient "not at absorbance maximum" refers to the extinction coefficient at 260 nm, which is the wavelength used to determine for optical density quantitation.
See the DECODED article about quantifying your oligo at: https://www.idtdna.com/pages/education/decoded/article/oligo-quantification-getting-it-right
Does IDT offer NED labeled oligos?
NED is an Applied Biosystems (Life Technologies) product. However, IDT can label oligos with ATTO 550, which is very similar to NED in both its excitation and emission spectra.
Depending on the machine you are using for identification, calibration may be needed, especially if you are performing multiplex qPCR.
Do you have any positively charged fluorophores?
No, all of the IDT fluorophores are negatively charged.
Do I resuspend fluorescently labeled oligos the same as I would standard, unlabeled oligos?
We strongly recommend resuspending and storing fluorescently labeled oligos in a buffered solution such as TE (10 mM Tris pH 8.0, 0.1 mM EDTA) at -20°C. This will help to increase the shelf life of your dye labeled oligos. Also, avoid unnecessary freeze thaw cycles by preparing a freezer stock solution (100 µM) and several working aliquots. Use the IDT Resuspension Calculator under the "SciTools®" tab of the IDT website for help determining volumes to use.
Can I use HEX, TET, and 6-FAM labeled primers in my multiplex qPCR?
Although HEX and TET are both chemical derivatives of Fluroescein, each has its own specific absorbance and emission wavelength characteristics. With properly calibrated equipment, each signal should be easily distinguishable.
If your equipment does not have dye specific filters or cannot be dialed in to a specific wavelength, then you should choose dyes with wavelength spectra that are further apart.
If your equipment does not have dye specific filters or cannot be dialed in to a specific wavelength, then you should choose dyes with wavelength spectra that are further apart.
Can I order an RNA oligo modified with fluorophore?
We offer a large variety of fluorophores available for both RNA and DNA. We can label the oligos at 5', 3', and internal positions. A list of fluorophores available can be found under Products > Modifications > Fluorophore.
Are there any special requirements for the handling and storage of my labeled probes?
Labeled probes should, for the most part, be handled as other custom oligonucleotide sequences. We recommend resuspending probes in TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA), such as or IDTE. Non-DEPC treated nuclease‑free water can be used, although it can limit shelf life. Ideally, for each resuspended probe, create a stock and several aliquots and store them all at –20°C. For long term oligo storage, temperature is the most important factor to consider.
For long term storage, whether oligos are dried down, or resuspended in water (non-DEPC treated) or TE buffer, it is optimal to store them frozen.
For room temperature or 4°C storage, oligos that are resuspended in TE buffer are more stable than dry oligos. Oligos that are stored in water are the least stable. At 4°C oligos stored under all of these conditions are stable for at least 60 weeks.
For long term storage, whether oligos are dried down, or resuspended in water (non-DEPC treated) or TE buffer, it is optimal to store them frozen.
For room temperature or 4°C storage, oligos that are resuspended in TE buffer are more stable than dry oligos. Oligos that are stored in water are the least stable. At 4°C oligos stored under all of these conditions are stable for at least 60 weeks.
References
- Cavazos-Elizondo D, Aguirre-Soto A. Photophysical Properties of Fluorescent Labels: A Meta-Analysis to Guide Probe Selection Amidst Challenges with Available Data. Anal. Sens. 2022;2(5). doi:10.1002/anse.202200004.
*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.
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