Amplification: ConsumablesAmplification Reagents and Plastics
Factors Impacting Gene Expression AnalysisRNA Isolation RNA integrity, purity, and yield■■ Genomic DNA contamination■■ Inh...
RNA Isolation■■   Kits are designed and formulated to assist     in the isolation of highly pure and intact RNA     from d...
RNA Isolation               Aurum™ Total RNA Fatty and Fibrous Tissue Kit                                                 ...
RNA Isolation                       iScript™ RT-qPCR Sample Preparation Reagent                                           ...
RNA Isolation               Selection Guide                                                                               ...
Reagents —Reverse Transcription■■   Formulated for efficient reverse transcription     across a broad linear dynamic range...
Reagents               Reverse Transcription — iScript™ Kit Selector                                                Reduce...
Reagents                       Two-Step Reverse Transcription Reagents                       iScript™ Advanced cDNA Synthe...
Reagents               Two-Step Reverse Transcription Reagents               iScript cDNA Synthesis Kit                   ...
Reagents                                     One-Step RT-qPCR Reagents                                     iScript™ One-St...
Reagents —Real-Time qPCR■■   Patented Sso7d fusion enzyme technology     delivers higher processivity and inhibitor     to...
Reagents                       Real-Time qPCR Reagents Selection Guide                                                    ...
Reagents               Real-Time qPCR — SYBR® Green/EvaGreen                SsoAdvanced™ SYBR® Green Supermix             ...
Reagents                       Real-Time qPCR — SYBR® Green/EvaGreen                       SsoFast™ EvaGreen® Supermix    ...
Reagents                     Real-Time qPCR — SYBR® Green/EvaGreen                      iQ™ SYBR® Green Supermix          ...
Reagents                         Real-Time qPCR — SYBR® Green/EvaGreen                         Precision Melt Supermix    ...
Reagents               Real-Time qPCR Probes               SsoFast™ Probes Supermix                                       ...
Reagents                       Real-Time qPCR Probes                       iQ™ Multiplex Powermix                         ...
Reagents               Standard PCR               iProof™ High-Fidelity DNA Polymerase                                    ...
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
Bio-Rad Amplification Reagents
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Bio-Rad Amplification Reagents

  1. 1. Amplification: ConsumablesAmplification Reagents and Plastics
  2. 2. Factors Impacting Gene Expression AnalysisRNA Isolation RNA integrity, purity, and yield■■ Genomic DNA contamination■■ Inhibitors of cDNA synthesis and qPCR■■ RNase and DNase contamination■■Reagents — Reverse Transcription cDNA synthesis efficiency■■ RNA protection■■ Input RNA capacity■■ Accurate representation of mRNA■■Reagents — Real-Time qPCR Detection sensitivity■■■■ Assay specificity■■ Inhibitors in sample■■ Reproducibility of thermal cycling conditions and instrument compatibilityPCR Plastic Consumables Instrument compatibility■■ Optimum performance■■ Automation friendly■■ Potential source of contamination and inhibition■■
  3. 3. RNA Isolation■■ Kits are designed and formulated to assist in the isolation of highly pure and intact RNA from different starting materials■■ RNA is compatible with a variety of downstream applications – Real-time qPCR – Northern blotting – Microarray analysis – cDNA library construction■■ DNase treatment ensures genomic DNA removal
  4. 4. RNA Isolation Aurum™ Total RNA Fatty and Fibrous Tissue Kit Aurum Total RNA 96 Kit RNA Isolation ■■ PCR-ready RNA in less than 60 min ■■ High-throughput total RNA isolation in less than 60 min ■■ PureZOL™ efficiently lyses cells and tissues, deproteinates RNA, and ■■ High yield of intact total RNA from a wide range of starting materials, inactivates endogenous nucleases in a single step including cultured cells, bacteria, and yeast, as well as plant and animal tissues ■■ Guanidine isothiocyanate and β-mercaptoethanol efficiently lyse samples ■■ High yield of intact total RNA from difficult-to-disrupt samples, including plant and quickly inactivate RNases and animal tissues ■■ RNase-free reagents and plastic consumables ensure the integrity of ■■ Well suited for fungal samples that are rich in RNases isolated RNA ■■ RNase-free reagents and plastic consumables ensure the integrity of ■■ Kit includes DNase I for removal of genomic DNA contamination isolated RNA ■■ Compatible with Aurum vacuum manifold ■■ Kit includes DNase I for removal of genomic DNA contamination For more information, request bulletin 2919. www.bio-rad.com/ ■■ Easy-to-use spin or vacuum protocol rna-isolation For more information, request bulletin 5282. Aurum Total RNA Mini Kit ■■ PCR-ready RNA in less than 60 min ■■ Guanidine isothiocyanate and β-mercaptoethanol efficiently lyse samples and quickly inactivate RNases ■■ High yield of intact total RNA from a wide range of starting materials, including cultured cells, bacteria, and yeast, as well as plant and animal tissues 732-6800, 2 x 96-well preps ■■ RNase-free reagents and plastic consumables ensure the integrity of Aurum Total RNA 96 Kit isolated RNA ■■ Kit includes DNase I for removal of genomic DNA contamination ■■ Easy-to-use spin or vacuum protocol For more information, request bulletin 2920. 732-6830, 50 preps 732-6820, 50 preps Aurum Total RNA Fatty and Fibrous Tissue Kit Aurum Total RNA Mini Kit© 2011 Bio-Rad Laboratories, Inc. Amplification Reagents and Plastics 5
  5. 5. RNA Isolation iScript™ RT-qPCR Sample Preparation Reagent PureZOL™ RNA Isolation Reagent ■■ Reagent stabilizes RNA and removes genomic DNA in less than 10 min ■■ Single-solution format permits recovery of RNA from small quantities of ■■ Suitable for adherent or suspension animal cells tissues or cells, making it ideally suited for gene expression studieswww.bio-rad.com/iscript ■■ RT-qPCR is directly enabled from cells without RNA purification when ■■ Efficient RNA purification from cultured cells, yeast, viruses, and bacteria, combined with an iScript reverse transcription kit and real-time supermix as well as plant and animal tissues ■■ Reagent allows multiplex real-time detection of up to 4 targets from as few ■■ PureZOL efficiently lyses cells and tissues, deproteinates RNA, and as 10 cells inactivates endogenous nucleases in a single step ■■ Ideal for rapid, high-throughput gene expression analysis ■■ Scalable starting sample amount For more information, request bulletin 5736. ■■ Convenient isolation of RNA, DNA, and protein from the same sample 104 Aurum™ Vacuum Manifold ■■ Vacuum-mediated nucleic acid purification platform ■■ Versatile manifold format adaptable for 96-well plate or up to 10 3 18 spin columns RFU ■■ Manifold ensures fast, high-quality sample preparation while maintaining the simplicity of vacuum processing 10 2 ■■ Unique vacuum regulator design allows for complete control of negative pressure 0 10 20 30 40 Cycles iScript RT-qPCR sample preparation reagent generates linear results over varying input cell amounts. HeLa cells (125, 25, 5, and 1 cells/µl) were treated and analyzed for GAPDH expression levels using iScript cDNA synthesis kit and iQ ™ SYBR ® Green supermix on the CFX96™ real-time PCR detection system. RFU, relative fluorescence units. 732-6890, 100 ml PureZOL RNA Isolation Reagent 170-8899, 5 x 10 ml 732-6470, 1 unit iScript RT-qPCR Sample Preparation Reagent Aurum Vacuum Manifold Amplification Reagents and Plastics 6 Visit us on the Web at www.bio-rad.com.
  6. 6. RNA Isolation Selection Guide Aurum Total RNA Kits PureZOL RNA RNA Isolation Mini Fatty and Fibrous Tissue 96 Isolation Reagent Format Mini column Mini column 96-well plate Single solution Filtration (vacuum or spin) Filtration (vacuum or spin) Filtration (vacuum or spin) organic extraction Maximum starting material amounts Cultured cells 2 x 106 1 x 107 1 x 106 1 x 107 Bacterial cells 2.4 x 109 2.4 x 109 8 x 108 2.4 x 109 Yeast cells 3 x 107 3 x 107 2 x 107 3 x 107 Hard animal tissue 20 mg 100 mg — 100 mg Soft to moderately 40 mg 100 mg — 100 mg hard animal tissue Plant tissue 40 mg 100 mg — 100 mg Isolation method Silica membrane Lysis with PureZOL reagent, Silica membrane Organic extraction purification on silica membrane Number of preps 50 mini preps 50 mini preps 2 x 96-well plate 50 or 100 (1 ml/prep) Number of washes 3 3 3 — DNase I included* Yes Yes Yes No DNase I digest time 15 min (animal tissue, 25 min) 15 min 10 min — Total preparation time** <50–80 min (with DNase I digest) <50–80 min (with DNase I digest) <60 min (with DNase I digest) <60 min Binding capacity >100 µg >100 µg >40 µg — Elution volume 2 x 40 µl 2 x 40 µl 80 µl 30–100 µl * Removal not required. ** Total preparation time will vary depending on the tissue or cell type and on which format is used (vacuum or spin). For sample-specific yield information, please visit www.bio-rad.com/rna-isolation and click the RNA Isolation Selection Guide. Ordering Information Catalog # Description Catalog # Description 732-6830 Aurum Total RNA Fatty and Fibrous Tissue Kit 170-8899 iScript RT-qPCR Sample Preparation Reagent, 500 reactions 732-6870* Aurum Total RNA Fatty and Fibrous Tissue Module 732-6880 PureZOL RNA Isolation Reagent, 50 ml 732-6820 Aurum Total RNA Mini Kit 732-6890 PureZOL RNA Isolation Reagent, 100 ml 732-6800 Aurum Total RNA 96 Kit 732-6470 Aurum Vacuum Manifold 170-8898 iScript RT-qPCR Sample Preparation Reagent, 100 reactions * Not provided with PureZOL RNA isolation reagent (see catalog #732-6890 or #732-6880 to order separately).© 2011 Bio-Rad Laboratories, Inc. Amplification Reagents and Plastics 7
  7. 7. Reagents —Reverse Transcription■■ Formulated for efficient reverse transcription across a broad linear dynamic range■■ Potent RNase A inhibitors protect RNA during setup and reverse transcription■■ Flexible input RNA capacity to suit different experimental needs■■ Optimized for gene expression analysis using real-time qPCR
  8. 8. Reagents Reverse Transcription — iScript™ Kit Selector Reduce Maximize data Fast and Select my One-step One-step pipetting from single easy to use own primers RT-qPCR with RT-qPCR for variability 20 l reaction SYBR® Green probes iScript reverse iScript advanced iScript cDNA iScript Select iScript™ one-step iScript one-step transcription supermix cDNA synthesis kit synthesis kit cDNA synthesis kit RT-PCR kit RT-PCR kit for probes for RT-qPCR for RT-qPCR with SYBR® Green Sensitivity 1 µg–100 fg 7.5 µg–100 fg 1 µg–100 fg 1 µg–1 pg 100 ng–1 pg 1 µg–1 pg total RNA total RNA total RNA total RNA total RNA total RNA Reagents iScript reverse iScript reverse iScript reverse iScript reverse iScript reverse transcriptase transcriptase transcriptase transcriptase transcriptase (for one-step RT-PCR) (for one-step RT-PCR) 5x iScript 5x iScript advanced 2x SYBR® Green RT supermix 5x iScript reaction mix RT-PCR reaction mix 2x probes RT-PCR Kit Contents reaction mix 5x iScript reaction mix (dNTPs, oligo[dT], (dNTPs, oligo[dT], (dNTPs and (dNTPs, iTaq™ DNA reaction mix (dNTPs, random primers, (dNTPs, oligo[dT], random primers, and polymerase, iTaq DNA polymerase, random primers, and buffer components) uorescein, buffer components, buffer components) and stabilizers) and iScript reverse buffer components) SYBR® Green I dye, transcriptase) and stabilizers) Oligo(dT), Forward and Forward and random primers, and reverse primers reverse primers gene-speci c primer for target gene and probe for (GSP) enhancer (not included) target gene solution (3 vials) (not included) cDNA ready in cDNA ready in 35 min cDNA ready in 40 min cDNA ready in RT-qPCR data in RT-qPCR data 40 min for qPCR for qPCR for qPCR 40–90 min for qPCR 60–90 min in 60 min© 2011 Bio-Rad Laboratories, Inc. Amplification Reagents and Plastics 9
  9. 9. Reagents Two-Step Reverse Transcription Reagents iScript™ Advanced cDNA Synthesis Kit for RT-qPCR iScript Reverse Transcription Supermix ■■Increased qPCR data throughput and cost effectiveness from a single 20 µl for RT-qPCRwww.bio-rad.com/ reverse transcription (RT) reaction ■■ 1-tube format for simple and fast setup, and reduced pipetting variabilityiscript ■■ Superior sensitivity and broad linear dynamic range for RT (7.5 µg–100 fg) ■■ Liquid format at –20°C offers superior stability and eliminates freeze/thaw cycle ■■ 2-tube kit (5x iScript reaction mix and iScript reverse transcriptase) for ease ■■ Superior sensitivity and broad linear dynamic range for RT (1 µg–100 fg) of use and reduced reaction setup time ■■ Optimized blend of oligo(dT) and random primers ensures complete and ■■ Optimized blend of oligo(dT) and random primers ensures complete and unbiased RNA sequence representation unbiased RNA sequence representation ■■ RNase H+ MMLV reverse transcriptase (preblended with RNase inhibitor) ■■ RNase H+ MMLV reverse transcriptase (preblended with RNase inhibitor) delivers high sensitivity for real-time RT-qPCR and eliminates additional delivers high sensitivity for real-time RT-qPCR and eliminates additional RNase H+ step RNase H+ step ■■ Potent blend of RNaseA inhibitor protects RNA during setup and RT ■■ Potent blend of RNaseA inhibitor protects RNA during setup and RT ■■ Short 40 min protocol allows fast qPCR data generation ■■ Short 35 min protocol allows fast qPCR data generation For more information, request bulletin 6031. For more information, request bulletin 6125. 35 104 — 1 µg RNA Average Cq SD CV, % — 100 ng 103 104 100 ng 21.35 0.123 0.576 30 — 10 ng RFU — 1 ng 100 pg 31.56 0.147 0.465 102 — 100 pg 25 — 10 pg RFU RFU — 1 pg Cq 0 10 20 Cycles 30 40 103 20 103 102 15 –2 –1 0 1 2 3 4 0 10 20 30 40 10 20 30 40 log starting quantity Cycles Cycles iScript advanced cDNA synthesis kit for RT-qPCR provides iScript reverse transcription supermix for RT-qPCR efficiently Excellent data reproducibility. PGK-1 mRNA (~160 bp), a gene that superior sensitivity and a broad linear dynamic range for reverse reverse transcribes RNA over a broad linear dynamic range encodes a glycolytic enzyme, was quantified using iScript reverse transcription. Total RNA (7.5 µg–1 pg) from HeLa cells was reverse for reliable gene expression analysis data. Different amounts of transcription supermix for RT-qPCR both with 100 ng () and 100 pg () transcribed using the iScript advanced cDNA synthesis kit for HeLa cell RNA (amounts shown in inset) were reverse transcribed of input RNA. For each input RNA, 48 individual RT reactions were RT-qPCR in a 20 µl reaction. A tenfold dilution of generated cDNA and one-tenth of the resulting cDNA was used as a template to performed and one-tenth of the resulting cDNA was used in the qPCR was used as template to amplify α-tubulin in a 10 µl qPCR reaction amplify β-actin gene (~90 bp) in 20 µl qPCR reactions with iQ™ reaction with SsoFast™ probes supermix. The gene expression analysis with iQ™ SYBR® Green supermix on a CFX384™ real-time PCR SYBR® Green supermix. Standard curve R 2 = 0.999, efficiency = data show excellent reproducibility both with high and low levels of input detection system. Efficiency = 90.7%, R2 = 0.999, slope = –3.57. Cq, 99.7%, slope = –3.33. RFU, relative fluorescence units. target mRNA. The ~10 Cq difference for the 1,000-fold dilution of RNA quantification cycle; RFU, relative fluorescence units. (100 ng–100 pg) demonstrates good reverse transcription efficiencies across different input RNAs. Cq, quantification cycle; RFU, relative fluorescence units. Amplification Reagents and Plastics 10 Visit us on the Web at www.bio-rad.com.
  10. 10. Reagents Two-Step Reverse Transcription Reagents iScript cDNA Synthesis Kit iScript Select cDNA Synthesis Kit ■■ 2-tube kit (5x iScript reaction mix and iScript reverse transcriptase) for ■■ 5-tube kit (random primers, oligo[dT], 5x iScript Select reaction mix, iScript ease of use and reduced reaction setup time reverse transcriptase, and gene-specific primer enhancer solution) www.bio-rad.com/ iscript ■■ Superior sensitivity and broad linear dynamic range for RT (1 µg–100 fg) ■■ Choice of priming strategy ■■ Optimized blend of oligo(dT) and random primers ensures complete and ■■ Reliable synthesis of long cDNA >6 kb in length unbiased RNA sequence representation ■■ Superior sensitivity and broad linear dynamic range for RT (1 µg–1 pg) ■■ RNase H+ MMLV reverse transcriptase (preblended with RNase inhibitor) For more information, request bulletin 2894. delivers high sensitivity for real-time RT-qPCR and eliminates additional RNase H+ step ■■ Potent blend of RNaseA inhibitor protects RNA during setup and RT ■■ Short 40 min protocol allows fast qPCR data generation For more information, request bulletin 2894. Reagents 1,000 10,000 — 1 µg — 100 ng 104 — 10 ng — 1 ng — 100 pg — 10 pg RFU RFU RFU 100 — 1 pg 1,000 103 10 102 100 0 5 10 15 20 25 30 35 40 45 0 10 20 30 40 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 Cycles Cycles Cycles The iScript cDNA synthesis kit performs across a broad range iScript reagents provide potent RNaseA inhibition. iScript iScript Select cDNA synthesis kit performs reliably over 6 orders of concentrations. Input RNA (amounts shown in inset) was reverse reagents for RT-qPCR include an optimum blend of RNaseA of magnitude using a gene-specific primer approach. Human transcribed, and the resulting cDNA was amplified using iQ™ SYBR® inhibitor for protecting RNA integrity. Reverse transcription was total RNA from 1 μg to 1 pg was reverse transcribed using the iScript Green supermix. Standard curve r2 = 0.998, efficiency = 96.5%. RFU, performed using 0.1 pg of input RNA with iScript reagent alone (), Select cDNA synthesis kit. One-tenth of the resulting cDNA was relative fluorescence units. spiked with RNaseA (), or spiked with RNaseA without the RNaseA used as a template to amplify β-actin gene with iQ™ SYBR® Green inhibitor included in the reaction (). 18S rRNA (~70 bp) was supermix. Standard curve r = 1.000, efficiency = 92.2%. RFU, relative amplified using iQ™ SYBR® Green supermix. A significant Cq delay fluorescence units. was observed when the reaction included RNaseA but no RNaseA inhibitor, which demonstrates potent RNaseA inhibition. RFU, relative fluorescence units.© 2011 Bio-Rad Laboratories, Inc. Amplification Reagents and Plastics 11
  11. 11. Reagents One-Step RT-qPCR Reagents iScript™ One-Step RT-PCR Kit with SYBR® Green Benefits of iScript one-step kits: ■■ For use on a broad range of real-time PCR instruments ■■ Provide powerful combination of iScript RNase H+ reverse transcriptasewww.bio-rad.com/iscript ■■ Extremely sensitive detection (100 ng–1 pg) of input RNA and antibody-mediated hot-start iTaq™ DNA polymerase Are ideal for rapid, high-throughput gene expression analysis iScript One-Step RT-PCR Kit for Probes ■■ ■■ For use with all types of hybridization probes, including dual-labeled ■■ Perform cDNA synthesis and qPCR in 1 tube, minimizing handling and oligonucleotide probes, FRET probes, and molecular beacons contamination risk ■■ Extremely sensitive detection (1 µg–1 pg) of input RNA For more information, request bulletin 3066. PCR baseline-subtracted curve fit, RFU PCR baseline-subtracted curve fit, RFU 10,000 1,000 1,000 100 100 10 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 Cycles Cycles iScript™ one-step RT-PCR kit with SYBR® Green provides high reproducibility and iScript one-step RT-PCR kit for probes delivers unparalleled results over an sensitivity across a broad range of concentrations. Reactions were performed in triplicate, extremely wide dynamic range. RNA (1 µg–100 fg) isolated from HeLa cells using along with no-template controls, using GAPDH primers and 100 ng–100 fg total HeLa RNA. the Aurum™ total RNA kit was reverse transcribed and amplified using primers to Reactions were carried out on the iCycler iQ® real-time detection system. Standard curve β-actin and a FAM-labeled detection probe. Each dilution was performed in triplicate r = 1.000, efficiency = 95%, slope = –3.47. RFU, relative fluorescence units. and RT-PCR was carried out on the iCycler iQ detection system. Standard curve r = 1.000, efficiency = 97.2%, slope = –3.39. RFU, relative fluorescence units. Ordering Information Catalog # Description $ Two-Step Reverse Transcription Reagents One-Step RT-qPCR Reagents 170-8842 iScript Advanced cDNA Synthesis Kit for RT-qPCR, 50 x 20 μl reactions 170-8892 iScript One-Step RT-PCR Kit with SYBR Green, 50 x 50 μl reactions 170-8843 iScript Advanced cDNA Synthesis Kit for RT-qPCR, 250 x 20 μl reactions 170-8893 iScript One-Step RT-PCR Kit with SYBR Green, 200 x 50 μl reactions 170-8890 iScript cDNA Synthesis Kit, 25 x 20 μl reactions 170-8894 iScript One-Step RT-PCR Kit for Probes, 50 x 50 μl reactions 170-8891 iScript cDNA Synthesis Kit, 100 x 20 μl reactions 170-8895 iScript One-Step RT-PCR Kit for Probes, 200 x 50 μl reactions 170-8840 iScript Reverse Transcription Supermix for RT-qPCR, 25 x 20 μl reactions 170-8841 iScript Reverse Transcription Supermix for RT-qPCR, 100 x 20 μl reactions 170-8896 iScript Select cDNA Synthesis Kit, 25 x 20 μl reactions 170-8897 iScript Select cDNA Synthesis Kit, 100 x 20 μl reactions Amplification Reagents and Plastics 12 Visit us on the Web at www.bio-rad.com.
  12. 12. Reagents —Real-Time qPCR■■ Patented Sso7d fusion enzyme technology delivers higher processivity and inhibitor tolerance■■ Antibody-mediated hot-start polymerases enable instant activation and higher specificity■■ Choice of fast, standard, or universal cycling conditions■■ Formulated for optimal performance on a variety of real-time instruments Reagents
  13. 13. Reagents Real-Time qPCR Reagents Selection Guide SYBR® Green / EvaGreen Supermixes Probes Supermixes One-Step Kits for RT-qPCR SsoAdvanced™ SsoFast™ iQ™ SsoFast iTaq™ Fast iTaq™ EpiQ™ SsoFast iQ iQ SsoFast iTaq Fast iTaq iScript™ iScript SYBR® EvaGreen® SYBR® EvaGreen SYBR® Green SYBR® Chromatin Probes Supermix Multiplex Probes Supermix Supermix One-Step One-Step Green Supermix Green Supermix Supermix with Green SYBR® Supermix Powermix Supermix with ROX with ROX RT-PCR Kit RT-PCR Kit Supermix Supermix with Low ROX Supermix Green with ROX with SYBR® for ProbesReal-Time PCR Instrument ROX with ROX Supermix GreenBio-RadCFX96™, CFX96 Touch™,CFX384™, CFX384 Touch™, CFX ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔Connect™iQ™, iQ™5, MyiQ™, MyiQ™2 ✔ ✔ ✔ ✔ ✔ ✔■ ✔ ✔ ✔MiniOpticon™, DNA EngineOpticon® I and II ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔Applied BiosystemsStepOne/StepOne Plus ◆ ◆ ◆ ◆ ◆ ✔ ✔ ◆ ◆ ◆ ✔ ◆ ✔ ◆ ◆7500, ViiA7 ✔ ✔ ✔ ✔7000, 7300, 7700, 7900HT ✔ ✔ ✔StratageneMx3000P, 3005P, 4000 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔EppendorfMastercycler eprealplex 2 or 4 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔QIAGEN/CorbettRotor-Gene 3000, 6000, Q ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔RocheLightCycler 480 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔LightCycler 1.0, 1.5, 2.0 ▲ ▲ ▲ ▲ ▲ ▲ ✔ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲Idaho TechnologyLightScanner HR-1 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔LightScanner 32 ▲ ▲ ▲ ▲ ▲ ▲ ✔ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲✔ Recommended for use as is ◆ ROX reference setting must be turned “off” ▲ BSA must be added according to instrument specifications Amplification Reagents and Plastics 14 Visit us on the Web at www.bio-rad.com.
  14. 14. Reagents Real-Time qPCR — SYBR® Green/EvaGreen SsoAdvanced™ SYBR® Green Supermix Sso7d Polymerase ■■ Robust formulation delivers performance across a broad range of standard and fast cycling conditions www.bio-rad.com/ supermixes ■■ Sso7d fusion polymerase and optimized buffer deliver fast reaction times via instant polymerase activation and rapid polymerization kinetics to generate exceptional qPCR results in less than 30 min ■■ Increased resistance to PCR inhibitors ensures maximum efficiency, sensitivity, and reproducibility, and allows increased fluorescence when compared to other SYBR® Green supermixes ■■ Advanced formulation tolerates a broad range of reaction conditions, primer concentrations, and temperature ranges The dsDNA binding protein, Sso7d, stabilizes the polymerase-template complex, increases processivity, and provides greater speed and reduced reaction times compared For more information, request bulletin 6136. to conventional DNA polymerases. Sso7d fusion polymerases are significantly more resistant to PCR inhibitors, making the SsoAdvanced and SsoFast supermixes ideal choices for challenging applications, such as direct qPCR, without the need for sample preparation. Reagents 105 104 105 38 700 36 600 34 –d(RFU)/dT 500 32 RFU Cq 400 30 300 104 103 104 28 200 26 100 RFU RFU RFU 0 –12 –11 –10 –9 –8 65 70 75 80 85 90 95 18 19 20 21 22 log starting quantity Temperature, °C Cycles 103 102 103 102 10 102 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 Cycles Cycles Cycles SsoAdvanced™ SYBR® Green supermix provides extreme SsoAdvanced™ SYBR® Green supermix demonstrates superior Exceptional reproducibility can be achieved with SsoAdvanced™ sensitivity in the detection of a single copy target gene. inhibitor tolerance. The ADAR gene was amplified from HeLa cDNA in SYBR® Green supermix. Efficient discrimination and reliable The cyclin gene was amplified and detected from fivefold serial the presence of water alone, or in the presence of a known PCR inhibitor, quantification can be obtained from a 1.33-fold serial dilution of input dilutions of 10 ng–80 pg (■) and 3.2 pg (■) human genomic DNA. Eagle’s minimal essential medium (EMEM) with fetal bovine serum (FBS; 0, template. The GAPDH gene was amplified from varying amounts of Cyclin efficiency = 103%, R2 = 1 (for 10 ng–80 pg). Inset shows 2.5, 5, 10, and 20%), added to SsoAdvanced™ SYBR® Green supermix (■) human genomic DNA (1 ng–136 pg). From left to right: 1 ng, 753 pg, the standard curve for the various dilutions. Cq, quantification or a traditional Taq DNA polymerase–based qPCR master mix (■). 565 pg, 425 pg, 320 pg, 240 pg, 181 pg, and 136 pg. GAPDH cycle; RFU, relative fluorescence units. SsoAdvanced™ SYBR® Green supermix showed quality amplification efficiency = 96.2%, R2 = 0.999. Inset is a magnified view showing in all reactions (EMEM with 20% FBS data shown), while the Taq DNA robust discrimination and reproducible amplification (six replicates for polymerase–based qPCR master mix failed to amplify in all EMEM with FBS each input amount). RFU, relative fluorescence units. combinations (shown in the inset melt curve). RFU, relative fluorescence units.© 2011 Bio-Rad Laboratories, Inc. Amplification Reagents and Plastics 15
  15. 15. Reagents Real-Time qPCR — SYBR® Green/EvaGreen SsoFast™ EvaGreen® Supermix SsoFast EvaGreen Supermix with Low ROX ■■ Inhibitor tolerance and enhanced processivity with Sso7d fusion polymerase ■■ Blended with ROX for optimal performance on Applied Biosystemswww.bio-rad.com/ standard and fast 7500 real-time PCR instruments ■■ Robust formulation ensures maximum efficiency, sensitivity,supermixes and reproducibility for qPCR For more information, request bulletin 5919. ■■ Instant polymerase activation and rapid polymerization kinetics deliver fast qPCR results For more information, request bulletin 5816. 104 100.000 6,000 900 800 5,000 700 10.000 600 500 RFU 4,000 400 103 RFU RFU ∆Rn 300 3,000 1.000 200 100 2,000 0 0 10 20 30 40 0.100 Cycles 1,000 102 0 0.010 0 10 20 30 40 0 10 20 30 40 4 8 12 16 20 24 28 32 36 40 Cycles Cycles Cycles The unique fusion polymerase in SsoFast EvaGreen supermix SsoFast EvaGreen supermix demonstrates superior inhibitor The unique fusion polymerase in SsoFast EvaGreen supermix delivers extreme speed and generates exceptional qPCR results tolerance and direct qPCR capability from cell culture. Results with low ROX generates exceptional qPCR results on the ABI in less than 30 min. Tenfold serial dilutions of 10 ng–100 ag cDNA show efficient amplification and detection of a spike-in control 7500 fast real-time PCR system. Tenfold serial dilutions of 10 ng–100 fg from human spleen were used in each 20 μl reaction to detect template in the absence (■) or presence (■) of conditioned tissue cDNA from human spleen were used in each 20 μl reaction to detect 18S rRNA. 18S rRNA efficiency = 101.8%, r = 0.997. Total qPCR run culture medium using SsoFast EvaGreen supermix. Inset shows a α-tubulin. α-tubulin efficiency = 105.8%, r = 0.996. Total qPCR run time = 29 min. RFU, relative fluorescence units. competitor’s standard qPCR reagent is able to amplify the spike-in time = 39 min (not including melt curve). control template only in the absence (■) vs. presence (■) of culture medium. RFU, relative fluorescence units. Amplification Reagents and Plastics 16 Visit us on the Web at www.bio-rad.com.
  16. 16. Reagents Real-Time qPCR — SYBR® Green/EvaGreen iQ™ SYBR® Green Supermix iTaq™ Fast SYBR® Green Supermix with ROX ■■ Analysis of low-, medium-, and high-abundance target genes with superior ■■ Developed and validated for Applied Biosystems standard and fast 7500 sensitivity and efficiency and Stratagene real-time PCR instruments www.bio-rad.com/ supermixes ® ■■ Formulated for maximum SYBR Green I stability and performance in a ■■ Compatible with standard and fast thermal cycling protocols wide variety of real-time PCR instruments For more information, request bulletin 5737. ■■ Antibody-mediated hot-start polymerase for quick activation and increased specificity iTaq™ SYBR® Green Supermix with ROX ■■ Developed and validated for Applied Biosystems 7000, 7300, 7700, For more information, request bulletin 2764. 7900, StepOne, and StepOne Plus real-time PCR instruments ■■ Increased specificity and reduced primer-dimers with antibody-mediated hot-start polymerase For more information, request bulletin 3065. Reagents 10.000 PCR baseline-subtracted curve fit, RFU 1,000 1.000 ∆Rn ∆Rn 1.000 100 0.100 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Cycles Cycles Cycles ™ ® iQ™ SYBR® Green supermix generates precise, quantitative results. iTaq™ fast SYBR® Green supermix with ROX generates linear iTaq SYBR Green supermix with ROX generates superior results A fivefold dilution series (50 ng–80 pg) of human genomic DNA was amplified results over 6 orders of magnitude on the ABI 7500 fast real-time on the ABI 7900HT system. Total RNA from HeLa cells was reverse using the supermix, primers, and a probe specific to the IL-1β gene. Triplicate PCR system. Tenfold serial dilutions of 100 ng–100 fg cDNA from transcribed using the iScript™ reverse transcription supermix for reactions at each concentration were amplified along with no-template HeLa total RNA were used in each 20 µl reaction designed to detect RT-qPCR. A tenfold dilution of generated cDNA (10 ng–100 fg) was controls on the iCycler iQ® real-time system. Standard curve r = 0.999, β-actin. Efficiency = 93.0%, r = 0.998. Total qPCR run time = 31 min. used as template to amplify the β-actin gene. Standard curve R2 = 0.999, efficiency = 97.6%, slope = –3.38. RFU, relative fluorescence units. efficiency = 95.7%, slope = –3.43.© 2011 Bio-Rad Laboratories, Inc. Amplification Reagents and Plastics 17
  17. 17. Reagents Real-Time qPCR — SYBR® Green/EvaGreen Precision Melt Supermix EpiQ™ Chromatin SYBR® Green Supermix ■■ Optimized formulation containing EvaGreen dye delivers robust PCR and ■■ Robust formulation delivers superior sensitivity and efficiency for qPCR fromwww.bio-rad.com/ high resolution melt (HRM) performance genomic DNA templatessupermixes ■■ Sensitive and effective discrimination of all 4 SNP classes across a broad ■■ Protocol is optimized for difficult real-time qPCR reactions for high GC amplicons range of amplicons ■■ Supermix contains fluorescein and ROX and is compatible with all real- ■■ Accurate detection of CpG methylation status for epigenetic studies time PCR instruments except Applied Biosystems 7000, 7300, 7700, and 7900 models (additional ROX can be added by ordering the dye ■■ Exceptional room temperature stability for high-throughput HRM studies separately, catalog #172-5858) ■■ Reliable performance on any HRM-capable thermal cycler Please refer to page 22 for more details about the EpiQ chromatin analysis kit. For more information, request bulletin 6137. For more information, request bulletin 6020. A B 0.20 0.02 0.0 –0.1 0.15 0.00 Difference RFU –0.2 Difference RFU Difference RFU 0.10 –0.02 –0.3 –0.04 0.05 –0.4 –0.06 –0.5 0.00 –0.08 –0.6 –0.05 78 79 80 81 82 83 75 76 77 78 79 80 74 76 78 80 82 84 Temperature, °C Temperature, °C Temperature, °C Precision melt supermix delivers robust HRM for single nucleotide polymorphisms (SNPs). Discrimination of class I Accurate methylation detection with precision melt supermix. Mixtures and IV SNP genotypes are shown in panels A and B, respectively. Class I (A to G substitution) and class IV (A to T substitution) SNP of methylated and unmethylated human genomic DNA of varying ratios were genotypes from mouse genomic DNA were analyzed using precision melt supermix. Wild type (■), heterozygote (■), and homozygous analyzed using HRM on a CFX384 real-time PCR detection system. Increasing mutant (■) are shown in the difference plots normalized to wild-type samples. HRM analysis was performed on a CFX384™ real-time PCR amounts of methylated DNA (■, 0%; ■, 2%; ■, 5%; ■, 50%; ■, 75%; ■, 95%; detection system and genotypes were automatically assigned by Precision Melt Analysis™ software. Amplification was carried out for 35 and ■, 100%) were analyzed for methylation of the human RARB2 gene. The cycles. Total run time including melt curve = 150 min. RFU, relative fluorescence units. genomic region contains 7 CpG sites and is 88 base pairs in length. Total run time including melt curve = 190 min. RFU, relative fluorescence units. Amplification Reagents and Plastics 18 Visit us on the Web at www.bio-rad.com.
  18. 18. Reagents Real-Time qPCR Probes SsoFast™ Probes Supermix iQ™ Supermix ■■ Robust, simultaneous detection of up to 2 different gene targets using ■■ Maximum efficiency and sensitivity for qPCR using fluorogenic probes fluorogenic probes ■■ Reliable amplification over a wide dynamic range of human genomic and www.bio-rad.com/ supermixes ■■ Compatible with fast or standard cycling protocols plasmid DNA concentrations ■■ Inhibitor-tolerant polymerase suitable for analyzing difficult samples ■■ Contains antibody-mediated hot-start iTaq™ DNA polymerase for quick For more information, request bulletin 5869. activation and increased specificity For more information, request bulletin 2764. SsoFast Probes Supermix with ROX ■■ Developed and validated for Applied Biosystems standard and fast 7900HT real-time PCR instruments For more information, request bulletin 6051. Reagents 105 104 Standard Curve 1,000 Cq 104 103 RFU RFU RFU log starting quantity 103 102 100 102 101 0 10 20 30 40 0 5 10 15 20 25 30 35 40 45 50 0 10 20 30 40 Cycles Cycles Cycles SsoFast probes supermix delivers superior results for gene Exceptional reproducibility can be achieved on the CFX384 iQ supermix provides sensitive real-time detection over 8 orders expression analysis of two targets on the CFX96™ real-time real-time PCR detection system with SsoFast probes supermix. of magnitude. Tenfold dilutions of a plasmid containing 109–10 copies PCR detection system, with no difference in detection of a low- Efficient discrimination and reliable quantification can be obtained from of the α-tubulin gene were amplified using iQ supermix and a FAM- expressing gene in duplex or singleplex. cDNA from human liver 1.33-fold serial dilutions of input template. The GAPDH gene was labeled hybridization probe for detection. Eight replicates at each (100 ng) was used in each 20 µl reaction. (■) HEX-labeled GAPDH probe amplified from varying amounts of HeLa cDNA (1 ng–102 pg). From concentration were amplified along with no-template controls on the duplex reaction; (■) Texas Red–labeled IL-2 probe duplex reaction; left to right: () 1 ng, 565 pg, 320 pg, 181 pg, and 102 pg; MyiQ™ real-time PCR detection system. Standard curve r = 0.999, (■) HEX-labeled GAPDH probe simplex reaction; (■) Texas Red–labeled () 752 pg, 425 pg, 240 pg, and 136 pg. GAPDH efficiency = efficiency = 98.2%, slope = –3.36. RFU, relative fluorescence units. IL-2 probe simplex reaction. Total qPCR run time = 38 min. RFU, relative 91.5%, R2 = 0.997. Inset shows the standard curve for the various fluorescence units. dilutions. Total qPCR run time = 50 min. Cq, quantification cycle; RFU, relative fluorescence units.© 2011 Bio-Rad Laboratories, Inc. Amplification Reagents and Plastics 19
  19. 19. Reagents Real-Time qPCR Probes iQ™ Multiplex Powermix iTaq™ Fast Supermix with ROX ■■ Robust supermix formulated for sensitive and efficient multiplex qPCR ■■ Developed and validated for Applied Biosystems 7500 standard and fastwww.bio-rad.com/ and Stratagene real-time PCR instrumentssupermixes ■■ Reliable quantification of up to 4 targets (expression levels can vary up to 106-fold between target genes) or up to 5 targets ■■ Compatible with standard and fast thermal cycling conditions ■■ Linearity over 6 orders of magnitude of input cDNA and 4 orders of ■■ Antibody-mediated iTaq DNA polymerase provided in a convenient magnitude of input genomic DNA 2x supermix ■■ Suitable for a wide variety of applications, including gene expression For more information, request bulletin 5737. analysis, SNP genotyping, SNP analysis, GMO detection, and viral load detection iTaq Supermix with ROX ■■ Developed and validated for Applied Biosystems 7000, 7300, 7700, 7900, For more information, request bulletin 5348. StepOne, and StepOne Plus real-time PCR instruments ■■ Sensitive and accurate detection over 6 orders of magnitude ■■ Hot-start iTaq DNA polymerase in optimized buffer for simplex and duplex qPCR For more information, request bulletin 3065. 10.000 PCR baseline-subtracted curve fit, RFU 1,000 1.000 ∆Rn ∆Rn 1.000 100 0.100 0.100 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 0 10 20 30 40 Cycles Cycles Cycles iQ multiplex powermix produces highly reliable qPCR results Robust duplex qPCR results with iTaq fast supermix with ROX on the iTaq supermix with ROX produces superior results on the ABI 7900HT for up to five targets in a single tube, with no difference in Applied Biosystems 7500 fast real-time PCR system. cDNA inputs: system. Total RNA from HeLa cells was reverse transcribed using the detection of a low-expressing gene in multiplex or singleplex. 100-fold serial dilutions of cDNA from the equivalent of 1 µg–1 pg total RNA iScript™ reverse transcription supermix for RT-qPCR. A tenfold dilution of One-tenth of a 1 µg cDNA synthesis reaction of human thymus total were used in each 20 µl reaction. FAM-labeled 18S rRNA probe duplex generated cDNA (100 ng–1 pg) was used as template to amplify the β-actin RNA was used in each 20 µl reaction. FAM-labeled β-actin probe (), reaction (), VIC-labeled beta-2-microglobulin (B2M) probe duplex gene with a FAM-labeled probe. Standard curve R2 = 0.999, efficiency = Cy5-labeled α-tubulin probe (), HEX-labeled GAPDH probe (), reaction (), VIC-labeled B2M probe singleplex reaction (). 18S rRNA 98.0%, slope = –3.38. TAMRA-labeled cyclophilin probe (), Texas Red–labeled IL-2 probe (). efficiency = 98.6%, r = 0.999; B2M efficiency = 98.0%, r = 0.998. Total RFU, relative fluorescence units. qPCR run time = 45 min. Amplification Reagents and Plastics 20 Visit us on the Web at www.bio-rad.com.
  20. 20. Reagents Standard PCR iProof™ High-Fidelity DNA Polymerase iTaq DNA Polymerase ■■ A high-fidelity DNA polymerase with 52-fold more accuracy than Taq ■■ Antibody-mediated hot-start DNA polymerase for quick 3 min activation DNA polymerase at 95°C www.bio-rad.com/ pcrreagents ■■ Unique Pyrococcus-like proofreading enzyme is fused to a dsDNA binding ■■ Polymerase prevents nonspecific amplification and primer-dimers in both protein, Sso7d PCR and real-time PCR applications ■■ Long and fast PCR applications — fragments up to 37 kb are amplified in For more information, request bulletin 2779. less time (15–30 sec/kb) and with less enzyme (0.25–1 U/reaction) ■■ Convenient 2x supermix is available for iProof polymerase and buffer for GC-rich templates For more information, request bulletin 5211. dNTP Mix ■■ Formulated for consistency and higher efficiency in PCR and real-time PCR ■■ Robust dNTP solution withstands multiple rounds of freeze-thawing and Reagents temperature cycling BAC DNA (37 kb) Human genomic DNA (28 kb) 35 min iProof high-fidelity DNA polymerase 1 kb 2 hr Common high-fidelity polymerase – 48 kb 2 hr Taq polymerase 15 22 28 Template length 32 37 – 23 kb 1.5 hr 10 20 22 25 7.9 8 kb 9.5 hr 5.5 hr 5 2 hr 20 min 3.8 15 kb 16.5 hr 2.9 (Failed amplification) Total protocol time iProof high-fidelity DNA polymerase demonstrates unrivaled speed, leading to dramatically iProof high-fidelity DNA polymerase amplifies long templates with high yields. Left, various shorter overall reaction times. The reaction protocol for iProof polymerase was compared to the fragments up to 37 kb in length were amplified from BAC DNA using a combined annealing/ recommended protocols for two competing polymerases. Each protocol was designed to amplify 1, extension step of 10 min per cycle and 30 U/ml of iProof polymerase. Right, various sequences up 8, and 15 kb products in 30 cycles. Reactions with iProof polymerase used a two-step protocol with a to 28.8 kb were amplified directly from human genomic DNA using 30 U/ml of iProof polymerase in combined annealing and extension step, while the other reactions used three-step protocols with the GC buffer with a combined annealing/extension time of 10 min per cycle. minimum recommended extension times. Overall reaction times include temperature ramping times.© 2011 Bio-Rad Laboratories, Inc. Amplification Reagents and Plastics 21

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