KiCqStart® Primers

Custom Primers
Black Hole Quencher®
Dual-Labeled Probes
Molecular Beacons
LightCycler® Probes
Scorpions® Probes
WellRED Primers

Primers and Fluorescent Probes

For Quantitative Real-Time PCR
and other Applications

bionucleics

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 bionucleics

Sigma® Life Science Custom Products

A Custom Solution for Every Project – Guaranteed

Predesigned and Custom Products

Sigma Life Science is recognized as the world’s leading supplier of custom oligonucleotides and peptide libraries for the global life science
research community. Originally founded in 1986 as Genosys Biotechnologies, Genosys was acquired by Sigma-Aldrich in 1998 to form
Sigma-Genosys. In 2005, Sigma-Aldrich acquired Proligo and its wide range of specialized DNA and siRNA products. Sigma-Genosys and
Sigma-Proligo harmonized product lines and continue to provide cutting-edge oligonucleotide technology, superior service and competitive
prices under the Sigma brand.

The Sigma Advantage Global Manufacturing

We continuously invest in our worldwide operations and believe it Sigma has oligonucleotide manufacturing sites around the globe,
is not just the quality of our products that sets us apart but also the in nine countries – Australia, Canada, Germany, India, Israel, Japan,
quality of our service and technical expertise. Singapore, UK and USA. Our global customers receive consistent
high-quality products.
The Sigma advantage includes:

• Our Commitment to Quality

• Outstanding Customer Service and Technical Support
• Global Manufacturing
Our Commitment to Quality
Quality is an integral part of our manufacturing process. Sigma
analyzes all oligonucleotides, including probes, by mass spectrometry,
ensuring the highest quality products. Complementary techniques,
such as analytical chromatography, are routinely used to verify Manufacturing

specifications are met. Our sizeable investment in state-of-the-art

analytical equipment provides industry-leading tools to develop and Expertise of Our Scientists
monitor our process. With more than 25 years of experience in oligonucleotide synthesis,
we have the expertise to create the most technically challenging
Our fluorescently-labeled probes are manufactured using a rigorous
custom biomolecules. Our scientists collaborate with researchers
process, including:
around the world and together develop novel custom products. We
• Purification by reverse phase and/or ion exchange chromatography meet customer specifications, no matter how complex.

• Electrospray mass spectral analysis Quantitative PCR Tools

• Quality control documentation Sigma is pleased to offer a variety of educational tools for quantitative
PCR users including:
• Amber packaging
Outstanding Customer Service and Technical Support
• Technical and troubleshooting guides
Our dedicated staff of highly-trained customer service specialists are
• PCR and qPCR technical manual
available via email or telephone to provide timely solutions to every • Webinar series
customer inquiry. Providing real-time status for orders, our customer
service teams demonstrate total commitment to customer satisfac-
• Workshops
tion. With an extensive staff of Molecular Biologists and Chemists, our
technical experts are prepared to assist researchers with experimental
design, application support and troubleshooting. Whether contacting
us via the web, email or telephone, Sigma customers are provided
with best-in-class service and support.

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 sigma.com/probes 3

The MIQE Guidelines

The potential applications for quantitative real-time PCR (qPCR) By Following the MIQE Guidelines You Will:
have increased exponentially since the first description (Higuchi,
1993). However, researchers have been frustrated by complications • Promote experimental transparency
such as contamination, insufficient amplification, low sensitivity and • Ensure consistency between laboratories
uncertainty about what constitutes a suitable statistical analysis. Until
recently, there has been a lack of consensus about how to handle
• Maintain the integrity of the scientific literature
these obstacles. Publish or bring your product to market faster and with confidence
by adhering to The MIQE Guidelines.
An international research team published The MIQE (pronounced
Mykee) Guidelines in 2009 to address the challenges of performing
dependable qPCR measurements. To learn more about MIQE and download the paper, visit
sigma.com/miqe
The MIQE Guidelines:
Issues addressed by MIQE include–

• Experimental design
• Sample preparation
• Nucleic acid extraction
• Reverse transcription
• qPCR target information
• qPCR oligonucleotides
• qPCR protocol
• qPCR validation
• Data analysis

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 bionucleics

Detection Chemistries
Sigma offers primers, probes and reagents to support all qPCR assays. There are several types of probe structures that can be used including:
• Dual-Labeled Probes
DNA-Binding Dyes and Probes
• Molecular Beacons
Quantitative real-time PCR (qPCR) relies on real-time detection of
amplification products as they are formed in the reaction. This • LightCycler® Probes
can be accomplished using non-specific DNA binding dyes or • Scorpions® Probes
sequence-specific probes. These techniques and their benefits Each probe type enables researchers to measure an increase in
are described below. fluorescent signal that corresponds to an increase in the copy number
A Theoretical qPCR Assay
of the desired amplicon. In addition to the increase in sensitivity that
is gained from using sequence-specific probes for detection, these
probes can also be labeled with different fluorescent reporter dyes,
[F] allowing detection of multiple targets within the same PCR reaction.
threshold
thr
Baseline de
detection limit Benefits and Challenges of Multiplex Reactions
The use of probes labeled with different reporter dyes allows the
simultaneous detection and quantification of multiple target genes in
a single (multiplex) reaction.
Cq Cq Cq Cq
Cycle # There are situations in which multiplex reactions are beneficial
including:
Figure 1. During a qPCR assay, the progress of the reaction is monitored by tracking the increase
in fluorescence from an associated reporter dye molecule. The number of cycles required to
reach a threshold level of detection is the quantification cycle (Cq). The lower the Cq the higher
• Limited Template Availability: The number of amplifications can
the initial concentration of target and vice versa. be maximized

Non-specific DNA Binding Dye Detection • Large Numbers of Samples: Reducing the number of reactions
leads to cost savings
SYBR® Green I binds non-specifically to double-stranded DNA.
Upon binding, the dye undergoes a conformational change resulting However, there are a variety of challenges when developing a multi-
in high fluorescent emission, allowing measurement of the total plex assay including:
amount of double-stranded product present in the reaction after
each amplification cycle.
• Complex Design: The degree of difficulty increases with the
number of targets to be detected
SYBR Green I detection is a popular option due to its low cost and • Optimization Reaction Conditions: All primer/probe sets need
ease of use. However, multiple double-stranded species that may be similar reaction kinetics and the same buffer, therefore target
present cannot be discriminated when using SYBR Green I. Within sensitivity may be reduced compared to similar singleplex reactions
any PCR, there is the potential for primer dimer formation and non-
specific amplification products. Therefore, melt curves must be used Predesigned vs. Custom Assays
after amplification for estimating the specificity of amplified products.
Predesigned assays offer convenience since no time or effort is
It may be difficult to obtain accurate quantification at low target
needed for primer and probe designs or deciding on oligonucleotide
concentrations.
specifications, such as the right manufacturing scale, purification,
For this reason, many researchers use SYBR Green I detection for yield, etc.
initial screening, proof-of-concept experiments or common
Sigma’s KiCqStart® Primers are ready-to-order (all specifications are
applications, such as gene expression analysis, and progress to
probe-based detection for greater assay sensitivity and/or for set), predesigned SYBR Green I Primers for RT-qPCR and are available
multiplex analysis. for a variety of species. They can be searched and ordered online at
sigma.com/ksprimers.
Probe-based Detection Sometimes, demanding assays require a greater level of attention
Probe-based detection methods rely on one or more fluorescently than can be found with predesigned assays. All Sigma’s detection
labeled probes that are positioned between the two PCR primers. chemistries including SYBR Green I Primers, Dual-Labeled Probes,
Because the probe is sequence specific, it will only detect the Molecular Beacons, LightCycler Probes, and Scorpions Probes can be
presence of a single amplicon within the reaction.
customized to your exact specifications.

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 sigma.com/probes 5

Assay Design
OligoArchitect™ Primer and Probe Design Solutions OligoArchitect Consultative
Sigma® is pleased to offer OligoArchitect for all of your primer For more complex and demanding applications, utilize our
and probe design requirements. OligoArchitect includes both consultative service to ensure the success of your assay. With
personal consultation from our expert, technical support scientists,
our complimentary online design tool and our unique
your request, including all sequences and data analysis, will be MIQE
consultative service.
compliant and returned to you within 24-48 hours. If required, you will
OligoArchitect Online also receive follow-up assay optimization, data analysis assistance and
troubleshooting support. Requests for consultative design service can
For routine needs, improve your assay with our OligoArchitect online be submitted at sigma.com/probedesignonline.
design tool powered by the industry standard Beacon Designer™
(PREMIER Biosoft). The user-friendly interface utilizes the latest Designs can be completed for traditional PCR or qPCR using the
algorithms, provides results in real time, supports templates up to following detection chemistries (Locked Nucleic Acid (LNA) can be
10,000 base pairs, and allows for the adjustment of input parameters included in probe and beacon designs):
such as homopolymer run/repeat maximum length, G/C clamp length • SYBR Green I Primers • LightCycler Probes
and maximum primer pair TM mismatch. • Dual-Labeled Probes • Scorpions Probes
Designs can be completed for traditional PCR or qPCR using the • Molecular Beacons
following detection chemistries (Locked Nucleic Acid® (LNA®) can be Designs can typically be completed for the following PCR and
included in probe and beacon designs): qPCR applications:
• SYBR® Green I Primers • LightCycler® Probes • Traditional PCR • Endpoint genotyping
• Dual-Labeled Probes • Scorpions® Probes • SNP detection • Methylation analysis
• Molecular Beacons • Allele discrimination • High-resolution melting analysis
Our online design tool can be used for the following applications: • Pathogen detection • Transcript detection across
• Traditional PCR • Viral load quantification • Multiplexing exon junctions

• SNP detection • Gene expression analysis • Viral load quantification • Haplotyping

• Allele discrimination • Gene copy determination • Gene expression analysis • Northern blotting

• Pathogen detection • Endpoint genotyping • Gene copy determination • Southern blotting

• Multiplexing Other applications:

All reported sequences, associated properties, and assay parameters •
FISH (fluorescence in situ hybridization)
are available for export to Excel and convenient email ordering. Visit Sigma’s consultative service uses Beacon Designer from PREMIER Biosoft
sigma.com/probedesignonline to try OligoArchitect Online. International. Blastn and mfold are used to determine assay specificity.

Bioinformatics Services
With recent growth in the volume and complexity of genomics and
proteomics research, it is necessary for scientists to be able to capture
and use this information. Sigma recognized this need and developed
state-of-the-art bioinformatics capabilities, including:

• A dedicated team of bioinformatics professionals with extensive

expertise in genomics and proteomics computing applications.
• An in-house system of hardware, software, tools and databases.
Both proprietary and commercial software packages allow greater
flexibility in addressing researcher needs.
• Microarray oligonucleotide design, AQUA™ Peptide design,
PEPscreen®: peptide library design, gene / transcript / proteomic
annotation, gene / protein function classification, microarray data
analysis and more.
• Rapid and secure completion of projects. Entire genome
oligonucleotide microarray designs are completed in less than
two weeks. All sequence information is retained in a secure
environment using internal Blast databases for searches.

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 bionucleics

Reaction Optimization
Proper optimization can increase the efficiency and sensitivity of a Improved Assay Sensitivity and Specificity
qPCR assay. Using Locked Nucleic Acid® (LNA®)
Primer and Probe Placement LNA can be incorporated into any of our qPCR probe types, providing
enhanced sensitivity and specificity for your assay. LNA is a novel type
In general, amplicons should be between 50-200 bases in length.
of nucleic acid analog containing a 2’-O, 4’-C methylene bridge. A
Shorter amplicons tend to be more tolerant of less than ideal reaction
conditions, improving the consistency of results. comparison of LNA to DNA is shown below:

When quantifying RNA targets, select primers spanning exon-exon

Base Base
junctions to avoid amplification of contaminating genomic DNA in HO HO
cDNA samples. O O

Primer and Probe Concentration O

Optimization of primer and probe concentrations can improve the LNAMonomer

Monomer DNAMonomer
Monomer
detection level of a particular amplicon by approximately 10 cycles LNA DNA
depending on the sequence. Since a 3 Cq difference in amplicon When used with any standard bases (A,C,G,T, or U), probes
detection indicates approximately a 10 fold difference in template synthesized using LNA have greater thermal stability than
concentration, this simple step can greatly improve both the accuracy
conventional DNA or RNA and therefore form a stronger bond
and sensitivity of the reaction.
with the complementary sequence.
Primer Optimization Improves Reaction Sensitivity
The introduction of LNA chemistry into a qPCR probe may result in
60 an increase in the TM of up to 8 °C per LNA monomer substitution
55
in medium salt conditions compared to a DNA fluorescent probe. It
50

45
is possible to optimize the TM level and the hybridization specificity
Fluorescence

40 through specific placement of the LNA base(s) in the probe design as

35

30
Optimal
primer
shown below.
concentration
25

20 Probe Sequence LNA Bases TM* ∆TM ∆TM/LNA

15

10
Sub-optimal
primer GTGATATGC 0 29 °C –– ––
concentration

44 °C 15 °C +5 °C
5
GTGATATGC 3
15 20 25 30 35 40
Cycle
GTGATATGC 9 64 °C 35 °C +3.9 °C
Figure 2. Primer optimization assay using Sigma SYBR® Green mastermix. Optimization of primers * TM of duplex between probe and its complementary sequence
for the human UBC gene. All reactions contain exactly the same template but varying primer Note: The bolded and underlined bases denote LNA base
concentrations. Assay highlights how variation in primer concentration has an impact on the
sensitivity of the assay. Platform: Rotor-Gene™ Corbett Research Ltd. Description of protocol
optimization is referenced: Nolan T, Hands RE, Bustin SA Quantification of mRNA using real-time Incorporation of LNA into your qPCR probe can improve performance
RT-PCR. Nature Protocols 2006; 1:1559-1582 of many assays, including:
• SNP Discrimination: The presence of a single base mismatch has
Buffer Conditions a greater destabilizing effect on the duplex formation between
Assay performance may also benefit from optimization of buffer a LNA fluorescent probe and its target nucleic acid than with a
components (particularly MgCl2) and the internal reference dye. conventional DNA fluorescent probe.
Optimizing the concentration of these components is especially • Multiplex Assays: Incorporation of LNA bases allows simpler T M
important when designing multiplex assays or singleplex assays in optimization, providing more flexibility in probe placement.
which design of an appropriate probe/primer combination proves to
be difficult. • Problematic Target Sequences: Shorter probes can be designed
to address traditionally problematic target sequences, such as
AT- or GC-rich regions, highly repetitive sequences or regions with
difficult secondary structure. Short regions of homology in aligned
sequences can also be targeted.

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 sigma.com/probes 7

Data Analysis
As your qPCR experiments grow in complexity and the data volume Next Steps:
becomes overwhelming, rely on our expertise to help you advance
your research.
• Purchase Sigma® qPCR probes
• Before beginning your experiment, we will evaluate and provide • Select desirable design and analysis options
recommendations for developing protocols. • Send your experimental protocols and raw data in .xls or .mdf file
format to the email address below, and you will be contacted by
• After completing your experiment, we will process your raw data one of our expert biostatisticians
into a comprehensive results report, including all statistical
analyses as well as publication-ready tables and graphs.
Design and Analysis Options:
• Evaluation or development of assay design
2

1.8
MDH • Identification of optimal reference genes
• Identification of marker gene combinations
FBP

1.6 ADH2
M-Value (Anti Log2)

HSP
1.4

1.2
PDC
PGK
SUC
• Quantification of unknown samples
• Determination of statistical significance
ADH1
TPI
1
MIG
ADH5
0.8 ADH3

0.6 CYC
ADH6
Benefits:
ADH4
0.4
IPPI PDA Bactin Sigma’s Biostatistics service will allow you to maintain your focus on
0.2 critical data-gathering activities while avoiding the need to purchase
0 expensive software and train personnel in time-consuming statistical
Genes
analyses. You will obtain results promptly so that you can publish or
Figure 3. Example reference gene selection that can be included with the results report. bring your product to market faster and with confidence.
To request service, send an email to biostatistics@sial.com

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 bionucleics

KiCqStart® Primers
KiCqStart Primers are predesigned SYBR® Green I Primers for two-step Guaranteed Yields
and one-step RT-qPCR. Available for a variety of species, the primer
Guaranteed Appx. No. Appx. No. Appx. No.
pairs can be easily searched and ordered online.
OD Yield of nmoles of μg of Reactions*
Choose KiCqStart Primers for: 3 14 90 1,500

• Gene expression analysis *Estimate based on 14 nmoles or 30 µg for 3 OD and 450 nM in a 20 uL reaction. Estimate is
based on an average sequence length of 21 bases.

• Validating MISSION® siRNA and shRNA knockdowns To learn more and order, visit
Benefits of Using KiCqStart Primers Include: sigma.com/ksprimers
• Developed with sophisticated bioinformatics tools and validated in Companion Products
silico to avoid off-target amplification and SNP loci (when possible)
Two-step RT-qPCR with KiCqStart Primers is easy with ReadyScript®
• MIQE compliant – sequences are provided at the time of shipment cDNA Synthesis Mix and KiCqStart SYBR Green qPCR ReadyMixes.
• Compatible with any thermal cycler ReadyScript cDNA Synthesis Mix for Fast and Easy
Preparation of Your cDNA Templates for qPCR
How KiCqStart Primers Work ReadyScript cDNA Synthesis Mix is a sensitive and easy-to-use
solution for two-step RT-qPCR. This 5X concentrated mix provides
KiCqStart Primers are PCR primers that work with SYBR Green I.
all necessary components (except RNA template) for first-strand
When free in solution with only ssDNA present, SYBR Green I emits
synthesis including: buffer, dNTPs, MgCl2, primers, RNase inhibitor
a low signal intensity. As the reaction progresses and the quantity
of dsDNA increases, more dye binds to the amplicons and hence, protein, ReadyScript reverse transcriptase and stabilizers. ReadyScript
signal intensity increases. The illustration below depicts is a RNase H(+) derivative of MMLV reverse transcriptase, optimized for
the mechanism. reliable cDNA synthesis over a wide dynamic range of input RNA.
Cat. No. Product Description
RDRT ReadyScript cDNA Synthesis Mix

KiCqStart SYBR Green qPCR ReadyMix™ for All Real-Time

1. Dye in solution emits 2. Emission of the PCR Instruments
low fluorescence fluorescence by binding
KiCqStart SYBR Green ReadyMixes for fast qPCR come in a ready-to-
use 2X formulation, which includes SYBR Green I dye, an antibody-
SYBR Green I cycles between an unbound (denaturation) and
mediated Hot-Start Taq DNA polymerase, dNTPs, MgCl2, and
a bound (annealing through extension) state as the reaction
progresses, and signal intensity increases as the quantity of proprietary buffers and stabilizers. Just add primers and template, and
amplicons increase in later cycles. you will discover that ReadyMixes offer the following features and
benefits:
• Speed – assay results in as little as 33 minutes
Product Features Include: • Quality – highly efficient and sensitive real-time PCR results
• Amount: 3 OD minimum yield • Ease of use – little to no optimization required
• Purification: RP cartridge
• Sequence Lengths: 18 – 24 bases Cat. No.
KCQS00
Product Description
KiCqStart SYBR Green qPCR ReadyMix
• Quality Control: 100% mass spectrometry KCQS01 KiCqStart SYBR Green qPCR ReadyMix, Low ROX™
• Format: Supplied dry, two oligos, one per tube KCQS02 KiCqStart SYBR Green qPCR ReadyMix with ROX
KCQS03 KiCqStart SYBR Green qPCR ReadyMix, iQ™

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 sigma.com/probes 9

Custom Primers
Forward and/or reverse primers are required for all custom probe Related Products
and beacon detection chemistries offered by Sigma. We offer a range of products when the focus is speed and simplicity.
Choose Custom Primers for: Easy Oligo:
• Dual-Labeled Probes
• Amount: 3 OD
• Molecular Beacons • Concentration: 100 µM
• LightCycler® Probes • Purification: Desalt
• Scorpions® Probes • Sequence Lengths: 15 – 30 bases
Benefits of Using Sigma’s Primers Include: • Quality Control: 100% mass spectrometry
• Free design support with OligoArchitect • Format: Supplied in solution
• Comprehensive technical support • Ships within 24 hours of order receipt
• Quality guaranteed Pure & Simple Primers:
Product Features Include: • Amount: 3 OD
• Scale: 0.025, 0.05, 0.2, 1.0, 10, and 15 µmole • Purification: Cartridge
• Purification: Desalt, Cartridge, HPLC, and PAGE • Sequence Lengths: 12 – 35 bases
• Sequence Lengths: 10-120 bases (inquire for longer lengths) • Quality Control: 100% mass spectrometry
• Modifications: Over 200 available, including dyes (fluorescent and • Format: Supplied dry
non-fluorescent)
• Ships within 24 hours of order receipt
• Quality Control: 100% mass spectrometry
• Format: Supplied dry or in solution – tubes, plates, and mixed plates To learn more and order, visit
(forward and reverse primers)
sigma.com/oligos
• WellRED Oligos are also available
Guaranteed Yields iScale™ Oligos

Appx. No.
• Larger quantities for high-throughput projects
Scale (µmole) Guaranteed OD Yield Appx. No. of μg of Reactions* • Quantity: 10, 25, 50, 100, 250, 500 mg (inquire for larger quantities)
0.025 3 96 600 • Purification: Desalt, RP-HPLC, IE-HPLC
0.05 5 160 1,000
0.2 12 384 2,500
• Sequence Lengths: 10 – 50 bases (inquire for longer lengths)
1.0 40 1,280 8,500 • Modifications: Over 200 available, including dyes (fluorescent and
10.0 400 12,800 85,000 non-fluorescent)
15.0 600 19,200 130,000 • Quality Control: 100% mass spectrometry
*Estimate based on 32 µg for 1 OD and 900 nM in a 25 µL reaction. Estimate is based on desalt
purification and an average sequence length of 21 bases. • Format: Supplied dry

To learn more and order, visit

sigma.com/iscaleoligos

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 bionucleics

Choosing the Right Custom Probe

There is a wide selection of possible custom probe types that can be used for qPCR, and each has advantages for different applications. The
summary below highlights the applications of commonly used custom probes (one red dot is good, two is better).

Application Reference Guide

SYBR® Green I Dual-Labeled Dual-Labeled Molecular LightCycler® Scorpions®
Primers Probes LNA® Probes Beacons* Probes* Probes*
APPLICATION
Mass screening ••
Microarray validation •• •
Multiple target genes / few samples • •
SNP detection •• • • ••
Allele discrimination •• • • ••
Pathogen detection • • •• • • ••
Multiplexing •• •• •• • ••
Viral load quantification • •• • • ••
Gene expression analysis • •• •• •• •• ••
Gene copy determination • •• • • ••
Endpoint genotyping •• ••
In vitro quantification or detection • ••
*LNA® can be incorporated into the probes for improved specificity.

Reporters, Quenchers and Instrument Compatibility Dye Substitutes

Modern qPCR platforms typically have multiple detection channels Several qPCR instruments utilize proprietary dyes which are not
enabling flexibility in the choice of probe labels. It is important to generally available commercially, such as VIC™ and NED™. When
select the fluorescent labels which are compatible with the detection seeking dye alternatives, the following criteria are important:
channels for the qPCR instrument and to ensure the correct filter • The excitation and detection wavelength are compatible with the
settings or detection calibration for the instrument. The Reporters instrument light source and detection system
and Instrument Compatibility table (see page 11) lists a selection of
some of the most widely used qPCR platforms and indicates which
• For probes, the quencher effectively absorbs light at the emission
wavelength of the reporter
fluorescent labels may be used. Please note that not all labels are
listed and many alternative reporters are available. For information on • The higher the extinction coefficient the brighter the dye, which
the use of non-standard labels with these platforms, contact your local contributes to sensitive detection
technical service professional (oligotechserv@sial.com). • When using multiple dyes (multiplex) the excitation and emission
wavelengths of each dye must be independent to avoid cross talk

Quenchers
Quenching molecules are typically placed at the 3’ end of single
molecule probes such as Dual-Labeled Probes, Molecular Beacons
and Scorpions Probes. Quenchers may be fluorescent (TAMRA™) or
nonfluorescent molecules (DABCYL, Black Hole Quencher® (BHQ®).
For optimal performance, the quencher’s absorbance spectrum
should match the reporter’s emission spectrum as closely as possible.
Recommended reporter/quencher combinations can be found in the
Spectral Properties table on page 11. To learn more about Black Hole
Quencher, see page 12.

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 sigma.com/probes 11

Spectral Properties Table

Dye Max. Max. Compatible Dye Max. Max. Compatible
EX (nm) EM (nm) Quencher EX (nm) EM (nm) Quencher
6-FAM™ 494 515 BHQ-1, TAMRA Cyanine 3 550 570 BHQ-2
JOE™ 520 548 BHQ-1, TAMRA Quasar® 570 3
548 566 BHQ-2
TET™ 521 536 BHQ-1, TAMRA Cal Fluor Red 590 4
565 588 BHQ-2
Cal Fluor® Gold 5401 522 541 BHQ-1 ROX™ 573 602 BHQ-2
HEX™ 2
535 555 BHQ-1, TAMRA Texas Red® 583 603 BHQ-2
Cal Fluor Orange 5602 540 561 BHQ-1 Cyanine 5 651 674 BHQ-3
TAMRA™ 555 576 BHQ-2 Quasar 670 5
647 667 BHQ-3
1
JOE/TET alternative 3
Cyanine 3 alternative 5
Cyanine 5 alternative Cyanine 5.5 675 694 BHQ-3
2
VIC® alternative 4
TAMRA alternative
Sigma® is a licensed supplier of a variety of dyes and quenchers and continually adds to its
portfolio of new chemistries. For assistance in the design of your probe and/or assays, visit
sigma.com/probedesignonline.

Reporters and Instrument Compatibility Table

Platform SYBR® FAM HEX JOE ROX TET Cyanine Cyanine TAMRA Texas Red LC Red 640 LC Red
Green I 3 5 705
ABI 7900HT • • • • • • •
ABI 7300 • • • • • •
ABI 7500 • • • • • • • • •
ABI 7700 • • • • • •
ABI 7000 • • • • •
ABI StepOne™ • • • • • • •
ABI StepOnePlus™ • • • • • • •
Bio-Rad iQ™ 5 • • • • • • • • •
Bio-Rad Opticon™ 2 • • • • •
Bio-Rad Chromo4™ • • • • • • • • • •
Bio-Rad MyiQ™ • •
Bio-Rad MiniOpticon™ • •
Bio-Rad CFX96™ • • • • • • • • • • • •
BIo-Rad SFX384™ • • • • • • • • • • • •
Agilent Mx4000® • • • • • • • • • •
Agilent Mx3000P® • • • • • • • • • •
Agilent Mx3005P® • • • • • • • • • •
Roche LightCycler® • • • •
Roche LightCycler 2 • • • • •
Roche LightCycler 480 • • • • • •
Cepheid SmartCycler® • • • • •
Cepheid SmartCycler II • • • • • •
Qiagen Rotor-Gene® 6000 • • • • • • • • •
Eppendorf
Mastercycler® ep realplex
• • • • • • •
Note: Not all qPCR instruments or reporters are listed.
Contact the instrument manufacturer for details on compatible reporters.

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 6-FAM

Abs (D
RFU (JO
bionucleics
250 300 350 400 450 500 550 600 650 700 750

Wavelength (nm)

Black Hole Quencher®

Two commonly used quenchers, TAMRA™ and DABYCL, limit the 6-FAM (518)
ultimate sensitivity and flexibility of qPCR. TAMRA is not a dark TET (538)
quencher and therefore contributes to an overall increase in T9 HEX (553)/JOE (554)
Cyanine 3 (565)
background because of its own native fluorescence. DABCYL, though TAMRA (583)
a dark quencher, has an inadequate absorption footprint that overlaps ROX (607)
LC Red (640)
very poorly with reporter dyes emitting above 480 nm (Figure 4).

Abs
BHQ-3 (672 nm) Cyanine 5
(667)
BHQ-2 (579 nm)
BHQ-1 (534 nm)

DABCYL-T9
RFU (JOE, TET, FAM)

T9
Abs (DABCYL-T9)

JOE 250 300 350 400 450 500 550 600 650 700 750
TET
Wavelength (nm)
6-FAM

Figure 5. Absorption spectra of the three BHQ variants (conjugated to T-9 and normalized to the
poly-T absorbance of 260 nm) with the emission maxima of the commonly-used reporters 6-FAM,
TET, HEX™, Cyanine 3, TAMRA, ROX™, LC Red 640, and Cyanine 5.

In addition to better spectral overlap, BHQ probes have much higher

250 300 350 400 450 500 550 600 650 700 750 signal-to-noise ratios when compared to the corresponding DABCYL
Wavelength (nm) or TAMRA probes (Figure 6).

Figure 4. Absorption spectra of DABCYL-T9 with the emission spectra of the commonly-used 5’-FAM Probes 5’-Cyanine 3 Probes
reporters 6-FAM™, TET™, and JOE™. DABCYL was normalized at the poly-T absorbance (260 nm).
9.0 80
RFU = relative fluorescence unit.
7.5
60
6-FAM (518)
Though DABYCL works well in MolecularTET Beacons
(538) (structured probes 6.0

with paired reporter and

T9quencher that utilize static quenching),
HEX (553)/JOE (554) 4.5 40
S:N

S:N
its absorption maximum of 474 nm places itCyanine
below 3 (565)
the
TAMRA (583)
maxima of 3.0

the reporters shown in Figure 4, therefore it is aROX

poor choice for
(607)
1.5
20

Dual-Labeled Probes (linear probes with separatedLCreporter

Red (640) and
Abs

BHQ-3 Cyanine 5 0.0 0

quencher that utilize FRET quenching).
(672 nm)
(667) TAM DAB BHQ-1 BHQ-2 DAB BHQ-1 BHQ-2
BHQ-2 (579 nm)
Black Hole QuencherBHQ-1
1 (BHQ®-1) (with an absorption maximum of 534
(534 nm) 3’ Quencher 3’ Quencher

nm) is absorbed at higher wavelengths than DABCYL and is directly

superimposable with the emission maxima of FAM, TET andProbes
5’-FAM JOE. This 5’-Cyanine 3 Probes 5’-Cyanine 5 Probes

provides a significant increase in both static

9.0 and FRET quenching 80 120

efficiencies, which makes it an excellent 7.5

quencher for Molecular
Beacons250 and 300 350 400 450 500 550 600 650 700 750
Dual-Labeled Probes. As shown in Figure 5, BHQ-1, 2, and 60 90
6.0
3 cover the spectrum from 480 Wavelength
nm into the (nm)
near IR making it possible
to utilize reporters that emit anywhere in4.5this range. 40 60
S:N

S:N

S:N

3.0
20 30
1.5

0.0 0 0
TAM DAB BHQ-1 BHQ-2 DAB BHQ-1 BHQ-2 DAB BHQ-2 BHQ-3

3’ Quencher 3’ Quencher 3’ Quencher

Figure 6. Signal-to-noise (S:N) ratios were calculated by dividing the fluorescence signal of a
25mer in the presence of a five-fold excess of an exact complementary target sequence by the
fluorescence intensity of the probe alone.

JB_82324_Primers and Fluorescent Probes Brochure.indd 12 9/4/14 8:26 AM

 sigma.com/probes 13

Finally, as shown in Figure 7, the BHQ variants readily permit single- In Summary, Black Hole Quencher:
tube multiplexing due to the increased variety of reporters that can 1) has no native fluorescence (emits heat instead of light), resulting
be effectively quenched with little or no cross-talk. This simplifies the in lower background fluorescence
design, implementation, and interpretation of multiplexed assays. 2) has increased signal-to-noise ratio, providing higher sensitivity
3) enables a wider choice of reporters for multiplexing

6-FAM
TET
Normalized Emission

JOE

450 500 550 600 650 700 750

Wavelength (nm)

6-FAM
TAM
Normalized Emission

ROX
CYANINE 5

450 500 550 600 650 700 750

Wavelength (nm)

Figure 7. The unique characteristics of the BHQ variants permit flexibility in the choice of
spectrally well-resolved reporters, which enable single-tube multiplexing with little or no
cross-talk.

JB_82324_Primers and Fluorescent Probes Brochure.indd 13 9/4/14 8:26 AM

 bionucleics

Dual-Labeled Probes
Dual-Labeled Probes are the most common probe type for qPCR and Product Features Include:
are often referred to as hydrolysis probes.
• Amounts: 1, 3, 5, and 10 OD
Choose Dual-Labeled Probes for: • Purification: HPLC
• Sequence Lengths: 15 - 40 bases
• Microarray validation • Multiplexing • Quality Control: 100% mass spectrometry
• Multiple target genes / • Viral load quantification • Format: Supplied dry in amber tubes
few samples • Gene expression analysis • Custom formats available (normalization, special plates, etc.)
• Pathogen detection • Gene copy determinations Sigma’s probes are provided in a format to simplify your experimental
planning.
Perfect for validating MISSION® siRNA and shRNA knockdowns
Guaranteed Yields
Benefits of Using Dual-Labeled Probes Include:
Guaranteed Appx. No. Appx. No. Appx. No.
• Design simplicity for sequence specificity OD Yield of nmoles of μg of Reactions*
• Extensive availability of reporter/quencher combinations 1 4 32 800
• Increased sensitivity 3 12 96 2,400
Add LNA® to Your Probe for: 5 20 160 4,000
• Increased thermal stability and hybridization specificity 10 40 320 8,000
• Greater accuracy in SNP detection, allele discrimination, and in vitro * Estimate is based on 4 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction
quantification or detection (5.0 pmol/reaction). Estimate is based on an average sequence length of 25 bases.

• Easier and more sensitive probe designs for problematic The most common reporter and quencher combinations are
target sequences listed below:
Spectral Properties Table
How Dual-Labeled Probes Work Dye Max. EX Max. EM Compatible
(nm) (nm) Quencher
A Dual-Labeled Probe is a single-stranded oligonucleotide
labeled with two different dyes. A reporter dye is located at 6-FAM™ 494 515 BHQ®-1, TAMRA™
the 5’ end and a quencher molecule located at the 3’ end. The JOE™ 520 548 BHQ-1, TAMRA
quencher molecule inhibits the natural fluorescence emission of
TET™ 521 536 BHQ-1, TAMRA
the reporter by fluorescence resonance energy transfer (FRET).
The illustration below depicts the mechanism. Cal Fluor® Gold 540 1
522 541 BHQ-1
HEX™2 535 555 BHQ-1, TAMRA
5’ Reporter Cal Fluor Orange 560 2
540 561 BHQ-1
R TAMRA™ 555 576 BHQ-2
5’ Reporter
3’ Quencher
Cyanine 3 550 570 BHQ-2
3’ Quencher
R Taq Quasar® 570 3
548 566 BHQ-2
Q DNA Q
polymerase Cal Fluor Red 5904 565 588 BHQ-2
Amplified Target DNA
ROX™ 573 602 BHQ-2
1. Probe in solution emits 2. Emission of the fluorescence
Texas Red® 583 603 BHQ-2
low fluorescence by hydrolysis Cyanine 5 651 674 BHQ-3
The primer is elongated by the polymerase and the probe binds Quasar 6705 647 667 BHQ-3
to the specific DNA template. Hydrolysis releases the reporter Cyanine 5.5 675 694 BHQ-3
from the probe/target hybrid, causing an increase in fluorescence. 1
JOE/TET alternative 3
Cyanine 3 alternative Cyanine 5 alternative
5

The measured fluorescence signal is directly proportional to the 2

VIC® alternative 4
TAMRA alternative
amount of target DNA.
Sigma® is a licensed supplier of a variety of dyes and quenchers and
continually adds to its portfolio of new chemistries. For assistance in
the design of your Dual-Labeled Probe assays, use our online design
tool at sigma.com/probedesignonline.

JB_82324_Primers and Fluorescent Probes Brochure.indd 14 9/4/14 8:26 AM

 sigma.com/probes 15

Molecular Beacons
Molecular Beacons are structured probes that are highly sensitive, Product Features Include:
sequence specific, and are used for sequence detection in qPCR and
• Amounts: 1, 3, 5, and 10 OD
in vitro studies.
• Purification: HPLC
Choose Molecular Beacons for: • Sequence Lengths: 15 - 40 bases
• Quality Control: 100% mass spectrometry
• SNP detection • Gene expression analysis • Format: Supplied dry in amber tubes
• Allele discrimination • Gene copy determination • Custom formats available (normalization, special plates, etc.)
• Pathogen detection • End point genotyping
• Multiplexing • In vitro quantification or Sigma’s probes are provided in a format to simplify your experimental
• Viral load quantification detection planning.

Benefits for Using Molecular Beacons Include: Guaranteed Yields

• Probe preserved during the reaction Guaranteed Appx. No. Appx. No. Appx. No.
• Increased specificity OD Yield of nmoles of μg of Reactions*
Add LNA to Your Probe for: 1 3 32 600

• Increased thermal stability and hybridization specificity 3 9 96 1,800

• Greater accuracy in SNP detection, allele discrimination, and in vitro 5 15 160 3,000
quantification or detection 10 30 320 6,000
• Easier and more sensitive probe designs for problematic target * Estimate is based on 3 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction (5.0 pmol/
sequences reaction). Estimate is based on an average sequence length of 30 bases.

The most common reporter and quencher combinations are

How Molecular Beacons Work
listed below:
A Molecular Beacon is a single-stranded bi-labeled fluorescent
probe held in a hairpin-loop conformation (around 20 to 25 nt) by Dye Max. EX Max. EM Compatible
complementary stem sequences (around 4 to 6 nt) at both ends (nm) (nm) Quencher
of the probe. The 5’ and 3’ ends of the probe contain a reporter 6-FAM™ 494 515 BHQ-1, DABCYL
and a quencher molecule, respectively. The loop is a single- Fluorescein 495 520 BHQ-1, DABCYL
stranded DNA sequence complementary to the target sequence.
JOE™ 520 548 BHQ-1, DABCYL
The close proximity of the reporter and quencher causes the
quenching of the natural fluorescence emission of the reporter. TET 521 536 BHQ-1, DABCYL
The structure and mechanism of a Molecular Beacon is shown HEX 535 555 BHQ-1, DABCYL
below. Cyanine 3 550 570 BHQ-2, DABCYL,
Loop
ROX™ 573 602 BHQ-2, DABCYL
Sequence
Loop Sequence Texas Red® 583 603 BHQ-2, DABCYL,
5’ Reporter
Stem 3’ Quencher
Cyanine 5 651 674 BHQ-3, DABCYL,
Sequence R Cyanine 5.5 675 694 BHQ-3, DABCYL,
Q

Q R
5’ Reporter Amplified Target DNA Sigma® is a licensed supplier of a variety of dyes and quenchers and
3’ Quencher
continually adds to its portfolio of new chemistries. For assistance in
1. Unbound beacon with 2. Bound beacon with the design of your Molecular Beacon assays, use our online design
quenched fluorescence unquenched fluorescence
tool at sigma.com/probedesignonline.
Molecular Beacons hybridize to their specific target sequence
causing the hairpin-loop structure to open and separate the 5’
end reporter from the 3’ end quencher. As the quencher is no
longer in proximity to the reporter, fluorescence emission takes
place. The measured fluorescence signal is directly proportional
to the amount of target DNA.

JB_82324_Primers and Fluorescent Probes Brochure.indd 15 9/4/14 8:26 AM

 bionucleics

LightCycler® Probes
LightCycler probes are highly sensitive and sequence-specific Add LNA® to Your Probe for:
fluorescent probes designed for use with the Roche LightCycler
• Increased thermal stability and hybridization specificity
instruments.
• Greater accuracy in SNP detection and allele discrimination
Choose LightCycler Probes for: • Easier and more sensitive probe designs for problematic
target sequences
• SNP detection • Viral load quantification Product Features Include:
• Allele discrimination • Gene expression analysis
• Pathogen detection • Gene copy determination • Amounts: 0.1, 0.25, 1.5, and 15 OD
• Multiplexing • Purification: HPLC
• Sequence Lengths: 15 - 40 bases
Several reporters are available and are suitable for multiplex analysis. • Quality Control: 100% mass spectrometry
• Format: Supplied dry in amber tubes
Benefit of Using LightCycler Probes include: • Custom formats available (normalization, special plates, etc.)
• Increased specificity Sigma’s probes are provided in a format to simplify your
• Probe preserved during the reaction experimental planning.
• Increased thermal stability and hybridization specificity
• Greater accuracy in SNP detection and allele discrimination Guaranteed Yields of LightCycler Probes
• Easier and more sensitive probe designs for problematic
target sequences Guaranteed Appx. No. Appx. No. Appx. No.
OD Yield of nmoles of μg of Reactions*
How LightCycler Probes Work 0.1 0.4 3.2 80
0.25 1 8 200
A LightCycler Probe system consists of a pair of single-stranded
fluorescent-labeled oligonucleotides. Oligo Probe 1 is labeled at 1.5 6 48 1,200
the 3’ end with a donor reporter and Oligo Probe 2 is labeled at 15 60 480 12,000
its 5’ end with one of a few available acceptor reporters. The free
* Estimate is based on 4 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction (5.0 pmol/
3’ hydroxyl group of Probe 2 must be blocked with a phosphate reaction). Estimate is based on an average sequence length of 25 bases.
group (P) to prevent DNA polymerase extension. There should
be a space of 1 to 5 nt to separate the two probes from each The recommended constructs for Oligo Probe 1 and Oligo Probe 2 are
other. The structures and mechanism of a LightCycler probe are listed in the tables below:
shown below.
3’ Donor Fluorophore (FD)
Labels and Modifications for LightCycler Probes
FD FD Oligo Probe 1
FA P

FRET
Oligo Probe 1 5’ Acceptor 3’ end Fluorescein
Fluorophore (FA)
FA P Amplified Target DNA
Oligo Probe 2*
Oligo Probe 2 3’ end Phosphate
5’ end LightCycler Red 610, 640, 670 and 705
1. Probes in solution emit 2. Emission through fluorescence
low fluorescence resonance energy transfer
*For enhanced discrimination, LNA can be incorporated into Probe 2

During the annealing step of qPCR, the PCR primers and the
LightCycler Probes hybridize to their specific target regions Sigma® is a licensed supplier of a variety of dyes and quenchers and
bringing the donor dye into close proximity to the acceptor dye. continually adds to its portfolio of new chemistries. For assistance in
When the donor dye is excited by light from the LightCycler the design of your LightCycler Probe assays, use our online design tool
instrument, energy is transferred by FRET from the donor to at sigma.com/probedesignonline.
the acceptor dye. The acceptor reporter’s emission wavelength
is detected. The increase in fluorescence signal is directly
proportional to the amount of target DNA.

JB_82324_Primers and Fluorescent Probes Brochure.indd 16 9/4/14 8:26 AM

 sigma.com/probes 17

Scorpions® Probes
Scorpions probes are highly sensitive, sequence specific, bi-labeled Add LNA to Your Probe for:
fluorescent probe/primer hybrids designed for qPCR.
• Increased thermal stability and hybridization specificity
Choose Scorpions for: • Greater accuracy in SNP detection and allele discrimination
• Easier and more sensitive probe designs for problematic
• SNP detection • Viral load quantification target sequences
• Allele discrimination • Gene expression analysis Product Features Include:
• Pathogen detection • Gene copy determination
• Multiplexing • Endpoint genotyping • Amounts: 1, 5, and 10 OD
• Purification: HPLC
Because the probe and primer are incorporated into a single
molecule, the reaction kinetics of this probe are extremely fast. The
• Sequence Lengths: 30 - 60 bases (Uni-Probe) and 15 - 45 bases
(Bi-Probe)
reaction leading to generation of a fluorescent signal is essentially
instantaneous and occurs prior to any competing side reactions. This
• Quality Control: 100% mass spectrometry
enables Scorpions Probes to provide stronger signals, shorter reaction
• Format: Supplied dry in amber tubes
times and better discrimination than other conventional bi-molecular • Custom formats available (normalization, special plates, etc.)
mechanisms. It also allows for more reliable probe design. Sigma’s probes are provided in a format to simplify your experimental
planning.
Benefits of Using Scorpions Probes Include:
Guaranteed Yields
• Increased specificity
• Fast amplicon detection Guaranteed Appx. No. Appx. No. Appx. No.
• Exceptional signal-to-noise (bi-probes typically yield a stronger OD Yield of nmoles of μg of Reactions*
signal when compared to uni-probes) 1 2 32 400
5 10 160 2,000
How Uni-Probe Scorpions Probes Work 10 20 320 4,000
* Estimate is based on 2 nmoles or 32 μg for 1 OD and 200 nM in a 25 µL reaction (5.0 pmol/
The Scorpions Uni-Probe consists of a single-stranded bi-labeled reaction). Estimate is based on an average sequence length of 50 bases (Uni-Probe).
fluorescent probe sequence held in a hairpin-loop conformation
with a 5’ end reporter and an internal quencher directly linked to The available reporter and quencher combinations are listed below.
the 5’ end of a PCR primer via a blocker. The blocker prevents the Scorpions Probes include a HEG (hexathylene glycol) blocker.
polymerase from extending the PCR primer.
Uni-Probe
Loop
Loop Blocker Sequence 5’ Reporter Internal Quencher
Sequence
Internal
Quencher 5’ Reporter Fluorescein, 6-FAM™, HEX™, TET™, TAMRA™, DABCYL dT,
Stem
Sequence JOE™, ROX™, Cyanine 3, Cyanine 5, Cyanine 5.5, BHQ®-1,
Q
Internal
R Texas Red®, Rhodamine, Rhodamine Green™, BHQ-2,
Quencher
R Q Rhodamine Red™, Oregon Green® 488, Oregon BHQ-3
PCR Primer
5’ Reporter
PCR Primer
Newly Synthesized Green 500, Oregon Green 514
DNA Strand
Blocker Target DNA Complementary Sequence
Bi-Probe
1. Quenching of the fluorescence 2. Emission of the fluorescence
5’ Reporter 3’ Quencher
At the beginning of the qPCR, the polymerase extends the PCR
Fluorescein, 6-FAM, HEX, TET, TAMRA, JOE, ROX, TAMRA, DABCYL,
primer and synthesizes the complementary strand of the specific
Cyanine 3, Cyanine 5, Cyanine 5.5, Texas Red, BHQ-1,
target sequence. During the next cycle, the hairpin-loop unfolds Rhodamine, Rhodamine Green, Rhodamine BHQ-2,
and the loop-region of the probe hybridizes intramolecularly to Red, Oregon Green 488, Oregon Green 500, BHQ-3
the newly synthesized target sequence. Now that the reporter Oregon Green 514
is no longer in close proximity to the quencher, fluorescence
emission may take place. The fluorescent signal is detected by the Sigma® is a licensed supplier of a variety of dyes and quenchers and
qPCR instrument and is directly proportional to the amount of continually adds to its portfolio of new chemistries. For assistance in
target DNA. the design of your Scorpions Probe assays, use our online design tool
at sigma.com/probedesignonline.
To learn about the bi-probe mechanism, please visit sigma.com/
probes, and click Learn More under Scorpions Probes.

JB_82324_Primers and Fluorescent Probes Brochure.indd 17 9/4/14 8:26 AM

 bionucleics

WellRED Primers
Sigma is licensed by Beckman Coulter, Inc., to supply WellRED Yields and Estimated Number of Assays
dye-labeled oligos designed for use with the CEQ™/GeXP Genetic
Analysis Systems and may be used for direct hybridization or in PCR Yield (OD) 1 2 4 5 10
amplification. WellRED dye-labeled oligos are DNA oligonucleotides Purification PAGE Cartridge Cartridge HPLC HPLC
labeled with WellRED dyes, D2, D3 or D4, at the 5’ end. Approx. yield 5 10 20 25 50
(nmol*)
Estimated assays 500-1,000 1,000- 2,000- 2,500- 5,000-
WellRED Specifications (reactions) 2,000 4,000 5,000 10,000
Yields Cartridge: 2 and 4 OD *Estimate 1 OD = 5 nmol = 30 µg, for a 20 base oligo
HPLC: 5, 10, 50 and 100 OD
PAGE: 1 OD
Purification Cartridge, HPLC, PAGE Spectral Properties of WellRED Dyes
Length 7 – 100 bases Excitation Emission Molar extinction
Backbone Phosphodiester WellRED Oligo maximum (nm) maximum (nm) coefficient
Format Supplied Dry D2-PA 750 770 170,000
Modifications D2-PA, D3-PA or D4-PA at the 5’ end D3-PA 685 706 224,000
Packaging 2 mL Opaque Tubes D4-PA 650 670 203,000
Quality Controls MALDI-TOF MS, Electrospray Ionization MS,
OD by UV Spectroscopy
To learn more and order, visit
Technical Datasheet & Includes yield, Tm, MW, and µg/OD
Labels Duplicate set of labels with each order sigma.com/wellred
Additional Services Aliquoting into tubes
MSQC traces available upon request
Shipping Schedule Shipped within 4 to 5 business days

Benefits of WellRED Dye Chemistry

• WellRED oligos use cyanine-based fluorescent dyes with high
extinction coefficients that absorb in the near-infrared region,
thereby reducing background noise from biological materials,
resulting in highly sensitive detection.
• The CEQ/GeXP Genetic Analysis Systems enable DNA samples to be
kept in linear form during analysis. The CEQ/GeXP Genetic Analysis
Systems’ online denaturation process prevents any reformation
of secondary structures, especially during a long run without any
manual steps.
• WellRED dye chemistry and pre-heating enable the CEQ/GeXP
Genetic Analysis Systems to resolve complex structures, where other
systems fail.

JB_82324_Primers and Fluorescent Probes Brochure.indd 18 9/4/14 8:26 AM

 sigma.com/probes 19

OligoEvaluator™
Sigma is pleased to offer OligoEvaluator, our online oligonucleotide OligoEvaluator™
sequence calculator.

Functionality
Module Features
Analysis Enter up to 10 sequences at a time, and the tool
returns values for all major physical properties, such
as molecular weight, melting temperature, secondary
structure, and primer dimer formation (secondary
structure and primer dimer formation information
provided in simple-to-interpret text format, e.g.
secondary structure--strong)
Resuspension Enter the quantity, desired concentration, and
molecular weight, and the tool returns the volume
of water or buffer needed to resuspend a dry
oligonucleotide
Dilution Enter the stock (resuspension) concentration, desired
final volume, and desired final concentration, and
the tool returns the volume of stock solution needed
for dilution

Advanced Features
• Review secondary structure analysis and primer dimer formation OligoEvaluator Input Inferface

to check for problematic folding

• Check for oligonucleotide sequence homology with BLAST All reported properties are available for export to a convenient Excel
template. Your OligoArchitect™ login can be used for OligoEvaluator.
Algorithm
To try OligoEvaluator, visit sigma.com/oligocalculator.
• Powered by PREMIER Biosoft

JB_82324_Primers and Fluorescent Probes Brochure.indd 19 9/4/14 8:26 AM

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©2014 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA and SIGMA-ALDRICH are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the
suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip. Where bio
begins, JumpStart and ReadyMix are trademarks of Sigma-Aldrich Co. LLC. PEPscreen® is a registered trademark of Sigma-Aldrich Co. LLC. KiCqStart is a registered trademark used under license. 5-FAM™, 6-FAM™ JOE™, TET™, HEX™, ROX™, TAMRA™, and VIC®
and are trademarks and registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. Inc. Molecular Beacons are sold under license from PHRI, City of New York, Inc. LightCycler® Probes sold under license from Roche
Diagnostics GmbH. Scorpions® Probes are licensed from DxS Ltd. SYBR® and Rhodamine Green™ are trademarks and registered trademarks of Life Technologies corporation. Alexa Fluor®, BODIPY®, Cascade Blue®, Marina Blue®, Oregon Green®, Pacific Blue™,
Rhodamine Red™-X, and Texas Red®-X are trademarks and registered trademarks of and provided under an intellectual property license from Life Technologies Corporation. Black Hole Quencher® (BHQ®) is a registered trademark of Biosearch Technologies,
Inc. CAL Fluor® and Quasar® are registered trademarks of Biosearch Technologies, Inc. Locked Nucleic Acid® (LNA®) oligonucleotides produced under license from Exiqon A/S. CEQ is a trademark of Beckman Coulter, Inc. Rotor-Gene® is a registered trademark
of Qiagen N.V. MasterCycler® is a registered trademark of Eppendorf, Inc. Opticon™ and Chromo4™ are trademarks of Bio-Rad Laboratories. Mx4000®, Mx3000P®, and Mx3005P® are registered trademarks of Agilent. SmartCycler® is a registered trademark of
Cepheid, Inc. For complete label license information, please visit sigma.com/oligolicenses.

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