Bio-Rad SsoAdvanced Universal SYBR Green Supermix User Manual
SsoAdvanced™ Universal
SYBR® Green Supermix
Instruction Manual
For use with SYBR® Green–based real-time PCR applications
on all real-time PCR instruments
Catalog # 172-5270
172-5271
172-5272
172-5 2 74
172-5275
Bio-Rad Technical Support
For help and technical advice, please contact the Bio-Rad Technical Support department. In the United
States, the Technical Support department is open Monday–Friday, 5:00 AM–5:00 PM, Pacific time.
http://www.bio-rad.com
Bio-Rad Laboratories
Life Science Research
2000 Alfred Nobel Drive
Hercules, CA 94547
Telephone: 510-741-1000
Telex : 335-358
Toll Fr e e: 1-800-4-BIORAD (1-800-424-6723)
Fax: 510-741-5800
Free Fax: 1-800-879-2289
Online technical support and worldwide contact information are available at www.consult.bio-rad.com.
Legal Notices
LightCycler is a trademark of Roche Diagnostics GmbH. Rotor-Gene is a trademark of Corbett Research. Mastercycler is a
trademark of Eppendorf AG. Mx is a trademark of Stratagene Corporation. Bioanalyzer is a trademark of Agilent Technologies.
FAM, StepOne, and StepOnePlus are trademarks of Applera Corporation.
SYBR is a trademark of Life Technologies Corp. Bio-Rad Laboratories, Inc. is licensed by Life Technologies Corporation to sell
reagents containing SYBR Green I for use in real-time PCR, for research purposes only.
Bio-Rad’s real-time thermal cyclers are licensed real-time thermal cyclers under Applera’s U.S. Patent Number 6,814,934 B1 for
use in research, human in vitro diagnostics, and all other fields except veterinary diagnostics.
These products are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG:
U.S. Patent Numbers 6,767,512 and 7,074,367.
NOTICE TO PURCHASER: LIMITED LICENSE
Use of this product is covered by one or more of the following U.S. patents and corresponding patent claims outside the U.S.:
5,804,375; 5,538,848; 5,723,591; 5,876,930; 6,030,787; and 6,258,569. The purchase of this product includes a limited,
non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s
own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including
without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly,
by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate
license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Copyright © 2013 by Bio-Rad Laboratories, Inc. All rights reserved.
|
Table of Contents
Sso7d Fusion Enzyme Technology iii
Educational Resources iv
Reagent Evaluation and Comparison Tutorials iv
Protocol 1
Sample Preparation Considerations 1
RNA Samples 1
RNA Integrity and Purity 1
DNA Samples 2
Plasmid Samples 2
Assay Design Considerations 3
Some Key Design Considerations 3
Procedure 4
Reaction Mix Preparation and Thermal Cycling Protocol 4
Real-Time PCR Validation for Gene Expression Experiments 5
Determining the Optimal Reference Gene 5
Determining the Dynamic Range of the Reverse Transcription Reaction 6
Determining the Real-Time PCR Performance Characteristics 9
Troubleshooting Guide 12
Ordering Information 21
SsoAdvanced™ Universal SYBR® Green Supermix Instruction Manual | i
SsoAdvanced™ Universal SYBR® Green Supermix
Catalog # Supermix Volume Kit Size
172-5270 2 ml (2 x 1 ml vials) 200 x 20 μl reactions
172-5271 5 ml (5 x 1 ml vials) 500 x 20 μl reactions
172-5272 10 ml (10 x 1 ml vials) 1,000 x 20 μl reactions
172-5274 25 ml (5 x 5 ml vials) 2,500 x 20 μl reactions
172-5275 50 ml (10 x 5 ml vials) 5,000 x 20 µl reactions
Shipping and Storage
The SsoAdvanced universal SYBR® Green supermix is shipped on dry ice. Upon receipt, the
supermix should be stored at –20ºC in a constant temperature freezer and protected from
light. When stored in these conditions, the supermix is guaranteed for one year. When stored
at 4ºC, the supermix is guaranteed for three months. To avoid excess freeze-thaw cycles, we
recommend preparing aliquots for storage.
Kit Contents
SsoAdvanced universal SYBR® Green supermix is a 2x concentrated, ready-to-use reaction
master mix optimized for dye-based real-time PCR on any real-time PCR instrument (ROXindependent and ROX-dependent). It contains antibody-mediated hot-start Sso7d fusion
polymerase, dNTPs, MgCl2, SYBR® Green I dye, enhancers, stabilizers, and a blend of passive
reference dyes (including ROX and fluorescein).
Instrument Compatibility
This supermix is compatible with all Bio-Rad and ROX-dependent Applied Biosystems real-time
PCR instruments, and with the Roche LightCycler LC480, Qiagen Rotor-Gene Q, Eppendorf
Mastercycler ep realplex, and Stratagene Mx real-time PCR systems.
Product Use Limitations
The SsoAdvanced universal SYBR® Green supermix is intended for research use only, and is
not intended for clinical or diagnostic use.
Technical Assistance
Bio-Rad Laboratories takes great pride in providing best-in-class technical support through
our online, telephone, and field support. To obtain support, please visit www.bio-rad.com, call
1.800.4.BIORAD, or contact your local field applications scientist.
Quality Control
SsoAdvanced universal SYBR® Green supermix demonstrates high PCR efficiency and linear
resolution over a wide linear dynamic range. Stringent specifications are maintained to ensure
lot-to-lot consistency. This product is free of detectable DNase and RNase activities.
SsoAdvanced™ Universal SYBR® Green Supermix Instruction Manualii |ii |
Sso7d Fusion Enzyme Technology
Bio-Rad introduced our next generation of real-time PCR supermixes using our patented
Sso7d fusion protein technology, delivering a reagent that provides effective performance
in a wide range of qPCR applications. The dsDNA-binding protein, Sso7d, stabilizes the
polymerase-template complex, increases processivity, and provides greater speed and
reduced reaction times compared to conventional DNA polymerases, without affecting PCR
sensitivity, efficiency, or reproducibility.
Key Features and Benefits
Fast qPCR results and high performance — the Sso7d fusion polymerase and optimized
buffer deliver fast reaction times via instant antibody hot-start polymerase activation and
rapid polymerization kinetics to generate exceptional qPCR results in less than 30 min
Minimal inhibition of PCR — the polymerase’s increased resistance to PCR inhibitors
ensures maximum efficiency, sensitivity, and reproducibility
Single copy detection — data illustrate high sensitivity with amplification and detection from
a single copy of target gene
Robust discrimination and reproducibility — efficient discrimination and reliable
quantification can be obtained from 1.33-fold serial dilutions of input template
GC-rich targets — ability to amplify targets where other Taq-based supermixes
may be challenged
| iiiSsoAdvanced™ Universal SYBR® Green Supermix Instruction Manual | iii
Educational Resources
Understanding the Basics
To learn more about similarities and differences between PCR and real-time PCR, understand
how SYBR® Green and probe-based chemistries function, and see how data are collected and
interpreted, please view our interactive tutorial Understanding Real-Time PCR.
Reagent Evaluation and Comparison Tutorials
Reverse Transcription
When comparing two different reverse transcription kits, often not all characteristics of the
reverse transcription (RT) reaction are tested. The end result is that a decision is made using
a limited set of data and criteria. The following protocol and exercise have been written in an
effort to create a more robust, reliable, and reproducible method of testing sensitivity, efficiency,
and other critical characteristics when comparing reagent providers for reverse transcription
kits. Reagent Comparison Guide for Real-Time PCR
To view an interactive tutorial and learn about reverse transcription chemistry, enzymes,
and priming methods, as well as how to perform a reagent comparison, please click here.
Understanding Reverse Transcription
Supermixes
When comparing two different supermixes, often not all characteristics of the PCR reaction
are tested. The end result is that a decision is made using a limited set of data and criteria.
The Reagent Comparison Guide for Real-Time PCR was written in an effort to create a more
robust, reliable, and reproducible method of testing sensitivity, efficiency, and other critical
assay characteristics when comparing reagent providers for use on real-time PCR systems.
To view an interactive tutorial and learn about supermix chemistry and enzymes, as well as how
to perform a reagent comparison, please click here. Understanding Real-Time PCR Supermixes
SsoAdvanced™ Universal SYBR® Green Supermix Instruction Manualiv |iv |
Protocol
This protocol is intended for use with SYBR® Green-based assays on all real-time PCR systems
using a broad range of cycling conditions, template and primer input concentrations, and fast or
standard run times.
Sample Preparation Considerations
RNA Samples
Isolate RNA using the appropriate method for the given sample type (Aurum™ total RNA mini
kit for cell lines, Aurum total RNA fatty and fibrous tissue kit for tissue samples)
Compare the expected yield to the actual yield to ensure the isolation method yielded the
appropriate RNA concentrations (5–30 pg per cell, 0.1–4 µg per mg of tissue). When the yield
is less than expected, this may lead to suboptimal qPCR data results, due to less than ideal
quality samples resulting from suboptimal sample prep workflow
When the RNA will be used for RT-qPCR, it is recommended that you treat the sample
with DNase to remove residual contaminating DNA. DNase treatment is also a good idea
when isolating RNA from tissues that are high in DNA, as the excess DNA may affect
downstream applications
Store the RNA in an appropriate solution
– 0.1 mM EDTA (in DEPC-treated ultrapure water)
– TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0)
Store the RNA at –80ºC in single-use aliquots
RNA Integrity and Purity
Use the Experion™ automated electrophoresis system or the Agilent Bioanalyzer to evaluate
the integrity of the RNA sample. When using multiple samples in the comparison, ensure that
the RQI/RIN numbers are similar to ensure accurate qPCR results
Use an agarose gel to assess RNA integrity if the above systems are not available. Apply the
same analysis concepts. High quality RNA will yield two clean peaks, 18s and 28s. Degraded
RNA will appear as a smear on the gel
To assess purity, evaluate the following spectrophotometer readings:
– A260/A280 >2.0 for pure RNA
– A260/A230 ~2.0 for pure RNA
• Lower ratios are indicative of contaminants from salts, carbohydrates, peptides, proteins,
phenols, and guanidine thiocyanate
SsoAdvanced™ Universal SYBR® Green Supermix Instruction Manual | 1
DNA Samples
Isolate DNA using the appropriate method for the given sample type (for example, column
purification for cell lines, phenol/chloroform or column purification for tissue samples)
Store the DNA in an appropriate solution
– 0.1 mM EDTA (in DEPC-treated ultrapure water)
– TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0)
Store the DNA at –80ºC in single-use aliquots
Assess DNA quality with an agarose gel; a single band indicates high integrity DNA, whereas
a smear indicates degraded DNA
Assess the DNA purity using a spectrophotometer for the following:
– A260/A230 >1.5 (lower ratios may be attributed to carryover guanidine, and/or inhibitors
– A260/A280 1.7–2.0 (lower ratios are indicative of contaminants from salts, carbohydrates,
– Higher ratios may be indicative of RNA contamination
Tips:
like humic acid and organics)
peptides, proteins, phenols, and guanidine thiocyanate)
Heat treating DNA may be required prior to qPCR to relax strong secondary structure
Using a restriction digest enzyme may be required for select qPCR applications, such as copy
number variation, to reduce signal-to-noise ratio.
Plasmid Samples
Prepare plasmids using an appropriate method
Store the stock plasmid in an appropriate solution
– TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
Store the plasmid at –80ºC in single-use aliquots
Assess plasmid quality with an agarose gel; a single band indicates high integrity plasmid,
whereas a smear indicates degraded plasmid or excess enzymatic activity
Assess the plasmid purity using a spectrophotometer for the following:
– A260/A280 1.7–1.9 (lower ratios are indicative of contaminants from salts, carbohydrates,
peptides, proteins, phenols, and guanidine thiocyanate)
– Higher ratios may be indicative of RNA contamination
2 | SsoAdvanced™ Universal SYBR® Green Supermix Instruction Manual2 |
Assay Design Considerations
When using custom designed assays, several important considerations should be noted:
Biological significance (correct isoform/splice variant chosen)
Sequence quality and secondary structure — evaluate using web-based tools to understand
the complexity of the structure, as it can impact the reaction performance
Sequence length — use the entire gene sequence, or a specific region of interest, to
optimally design an assay
Sequence masking — use web-based masking tools to mask low complexity and repetitive
regions to avoid assay design in these regions
Uniqueness of the sequence — use BLAST or BLAT to ensure no homology exists and help
avoid mispriming events
Uniqueness of the assay — use in silico PCR, or Primer-BLAST, to “blast” the primers against
the genome of interest to validate primer design specificity
Default settings in the software — ensure they are set correctly (for example, salt conditions,
oligo and amplicon sizes). The SsoAdvanced™ universal SYBR® Green supermix and the
qPCR cycling protocols have been optimized for assays with a primer melting temperature
(Tm) of 60ºC designed using the open source Primer3, Primer3Plus or Primer-BLAST, default
settings. For assays designed using other tools, the primer T
Primer3. Suggested settings: 50 mM Na+, 3 mM Mg++, 1.2 mM dNTPs, 250 nM annealing
oligo, SantaLucia/SantaLucia
should be recalculated using
m
Some Key Design Considerations
For optimal PCR efficiency, design the amplicon size between 70 and 150 bp (<70 bp may be
needed for degraded/FFPE templates)
Maintain primer lengths between 18 and 22 bp for good specificity and binding abilities
Annealing temperatures between 58 and 62ºC are optimal (greater range can be obtained
using Bio-Rad’s Sso7d-based supermixes); temperatures >60ºC may result in less binding
efficiency and <58ºC may result in less specificity
The optimal amplicon GC content should be within 40–60% (greater range can be obtained
using Bio-Rad’s Sso7d-based supermixes)
Avoiding primer secondary structures reduces potential primer-dimer issues
Avoid mispriming by ensuring there are no more than 2 Gs or Cs in the last 5 bases on the 3'
end of the primer
Design your assay such that at least one primer spans an exon:exon junction site to avoid
gDNA amplification
Alternatively, design the assay such that the primers are in separate exons and the intron
size is >1 kb
Tips to Get Started:
Always evaluate the performance of the supermix following the recommended reaction and
cycling conditions prior to modification
Be sure to set the activation time to 30 sec for cDNA and 2–3 min for genomic DNA
The 2x supermix has been optimized for 20 µl reactions in 96-well plates and 10 µl reactions
in 384-well plates
| 3SsoAdvanced™ Universal SYBR® Green Supermix Instruction Manual | 3
Loading...