Thermo Scientific PCR Reagent Selection Guide

Transcription

1 Thermo Scientific PCR Reagent Selection Guide

2 PCR Simplified Your work is challenging. Choosing the right PCR reagent shouldn t be. Standard High Yield Hot Start High Fidelity Long Range RT-PCR The Thermo Scientific PCR reagent line is a concise portfolio of powerful enzymes built to satisfy the broad range of demanding PCR applications. Whether you are performing routine detection or site-directed mutagenesis, our line provides a clear solution for success with your application. To deliver the ultimate in assay flexibility, our enzymes are supported by a range of convenient formats and custom services to suit any workflow. This complete portfolio of PCR solutions is the result of over years of ongoing development activities, which include both in-house research and collaborations with industrial and academic partners. Our commitment to continual innovation enables us to provide you with the best PCR systems, now and in the future. Look to the Thermo Scientific reagent line for a clear solution to your PCR challenge

3 Thermo Scientific PCR Enzyme Portfolio Standard PCR High Yield PCR Hot-Start PCR High Fidelity PCR Long Range PCR RT-PCR Application Guide: Achieve the best results by choosing an enzyme system designed for success with your application. Routine PCR applications requiring consistent and reliable Taq performance. Yes/No sequence detection. PCR applications requiring increased yield and sensitivity, to produce large amounts of PCR product. Challenging PCR applications requiring maximum specificity and sensitivity, to detect rare or complex targets. PCR applications requiring highly accurate amplification, to produce accurate PCR products with minimal sequence errors. PCR applications requiring robust, accurate amplification of target sequences greater than 5 kb in length. PCR applications that target RNA templates and require reverse transcription prior to PCR amplification. : 7 : 8 : 10 : 12 : 14 : 16 Taq DNA Polymerase ThermoPrime Taq DNA Polymerase Thermo-Start Taq DNA Polymerase Verbatim High Fidelity DNA Polymerase Extensor Long Range Enzyme Blend Verso Reverse Transcriptase Applications Routine PCR Sequencing Multiplexing Genotyping Library Construction Cloning 1 Mutagenesis Long PCR Pathogen Detection Single-cell PCR Specifications GC Rich Templates (w/ High Perf. Buffer) (w/ GC Buffer) (w/ Buffer 2) Low Copy Templates Long Templates Amplicon Size 5 kb 5 kb 5 kb 10 kb kb (w/ Buffer 2) Proofreading Activity Blunt or 3 -A Ends 3 -A 3 -A 3 -A Blunt 1 3 -A 2-Step RT-PCR Used to create a complete cdna pool. Aliquots from this pool can be used in separate PCR assays to study multiple genes. Verso cdna Synthesis Kit can be paired with any PCR Kit for complete 2-Step RT-PCR. 1-Step RT-PCR Reverse transcription and amplification steps are carried out in a single reaction tube. Convenient, fast analysis for studying single gene targets. Verso 1-Step RT-PCR Kits are available in two formats: High Yield (w/ ThermoPrime) and Hot-Start (w/ Thermo-Start) Performance Yield Specificity Sensitivity Accuracy / Fidelity 1 Enzyme creates blunt ends, A overhangs must be generated prior to ligation into T/A vector Legend Recommended Enzyme Suitable Alternative & Custom Services

4 Format Options Standard PCR Thermo Scientific PCR Enzyme Systems are available in a range of formats and pack sizes to suit any workflow. ENZYME KIT DNA Polymerase / 10X PCR Buffer / COMPLETE PCR KIT DNA Polymerase / 10X PCR Buffer / / mm MASTER MIX PCR Buffer containing DNA Polymerase, and Enzyme Kits are the most basic format. Kits include the enzyme, PCR buffer, and 25 mm solution, each in separate vials. must be purchased separately. Complete PCR Kits include all the components needed to perform PCR including an HPLC-purified dntp mix. Complete PCR kits are a convenient option that still allows for optimization. Master Mix formats consist of a single master buffer containing all the components needed for PCR. Master Mixes are the most convenient format and significantly reduce assay set-up steps. ENZYME KIT WITH REDDYMIX DNA Polymerase / 10X ReddyMix Buffer / COMPLETE PCR KIT WITH REDDYMIX DNA Polymerase/ 10X ReddyMix Buffer / / mm REDDYMIX MASTER MIX ReddyMix Master Mix containing DNA Polymerase, and Save time and effort with ReddyMix Versions! ReddyMix buffers contain an inert red dye that eases PCR set-up, and a gel loading precipitant that allows for direct loading onto agarose gels. The red dye aids in error free pipetting, and the precipitant allows the completed PCR reaction to be directly loaded onto agarose gels eliminating the need for separate gel loading buffer. ReddyMix formats ease reaction set-up and reduce handling time leading to faster results and improved reproducibility. For the ultimate in convenience and simplified assay setup, choose ReddyMix Master Mix (see figure below). Taq DNA Polymerase PCR is a highly sensitive reaction, and impurities in the enzyme mix can cause reduced efficiency or complete reaction inhibition. Any PCR application, no matter how routine, requires a Taq polymerase that is highly purified, and performs consistently from batch to batch. Thermo Scientific Taq DNA Polymerase is a 94 kda ultra-pure recombinant thermo-stable DNA polymerase obtained by high level expression of the Taq DNA polymerase gene in Escherichia coli. Thermo Scientific Taq DNA Polymerase is licensed and optimized for PCR and is able to amplify DNA fragments up to 5 kb. Stringent quality controls make this a highly reliable Taq DNA polymerase for use in all routine PCR and RT-PCR applications. Figure 1: Thermo Scientific Taq delivers successful amplification over a range of target lengths PCR amplification of 6 target lengths of human genomic DNA (76 bp, 295 bp, 653 bp, 800 bp, 2.2 kb, and 5 kb) each using 12.5 ng of genomic DNA template. bp kb Genomic DNA Target length >> Choose Your Preferred Format: ENZYME KIT DNA Polymerase / 10X PCR Buffer / Reliable performance in all routine PCR applications Extensive QC testing ensures batch-to-batch consistency COMPLETE PCR KIT DNA Polymerase / 10X PCR Buffer / / mm Standard PCR Kit VS ReddyMix Master Mix Standard PCR KIT REDDYMIX MASTER MIX Dispense Master Mix, Template and Primers Calculate and mix components to prepare master buffer Run PCR Protocol Choose ReddyMix Master Mix format for the most streamlined PCR workflow Dispense Master Mix, Template and Primers Load Gel & Analyze Run PCR Protocol Add gel loading buffer to each sample Custom Aliquoting Services To meet the demands of highthroughput applications, our PCR reagents can be supplied pre-aliquoted into ABgene PCR plastics, eliminating PCR set-up steps and allowing hundreds of reactions to be prepared in minutes. Load Gel & Analyze Notes Concentration: All buffers premixed with will be at 1.5 mm concentration in the final reaction. Unit Definition: One unit of enzyme is defined as the amount that will incorporate 10 nmoles of into acid insoluble material in 30 minutes at 74 C under the following analysis conditions: 25 mm TAPS, ph 9.3 (at 25 C) 50 mm KCl 2 mm 1 mm β-mercaptoethanol 250 μm of each: dctp, dgtp, dttp 250 μm [3H] datp (0.05 Ci/mmol) 1.25 μg/μl activated salmon sperm DNA Detailed Product Information For more information or to download the product manual, please visit: Units Reactions AB-0192/A 250 U 400 x 25 μl AB-0192/B 2500 U 4000 x 25 μl w/ AB-1192/A 250 U 400 x 25 μl AB-1192/B 2500 U 4000 x 25 μl Units Reactions AB-0192/A/N 250 U 400 x 25 μl AB-0192/B/N 2500 U 4000 x 25 μl w/ AB-1192/A/N 250 U 400 x 25 μl AB-1192/B/N 2500 U 4000 x 25 μl

5 High Yield PCR ThermoPrime Taq DNA Polymerase Figure 2: ThermoPrime Taq DNA Polymerase delivers high yield over a range of template concentrations. Figure 3: Easily detect pipetting errors with Red Hot Taq formats and ReddyMix kits and master mixes. PCR assays using standard Taq DNA polymerase and basic buffer formulations are subject to a loss in polymerase activity over the multiple high temperature steps of PCR cycling. This incremental loss of activity can result in decreased end yield and reduced assay sensitivity. ThermoPrime Taq DNA Polymerase exhibits enhanced thermal stability giving extended polymerase activity over standard Taq DNA polymerase formulations. A proprietary buffer component protects ThermoPrime during high temperature steps, ensuring optimal performance throughout the PCR reaction, leading to increased end yields. Sustained enzyme activity also helps maintain reaction efficiency and exponential amplification, making ThermoPrime an ideal enzyme for comparative analysis of gene expression. Enhanced enzyme stability for increased yield Advanced buffering system minimizes PCR optimization steps Convenient Master Mix formats PCR amplification of 8 concentrations of human genomic DNA (100, 50, 25, 12.5, 6.3, 3.2, 1.6 and 0.8 ng) using a 800 bp β-actin gene fragment as the target. Genomic DNA Template (ng) >> bp >> Red Hot formats include an inert red indicator dye in the enzyme mix, and ReddyMix kits contain an inert red dye in the buffer (in addition to a gel loading precipitant). THERMOPRIME Choose Your Preferred Format: AB-0301/A AB-0301/B ENZYME KIT DNA Polymerase / 10X PCR Buffer / w/ AB-1301/A AB-1301/B Red Hot Taq Version (Red Dye w/enzyme) AB-0406/A AB-0406/B w/ AB-1406/A AB-1406/B AB-0301/A/N AB-0301/B/N COMPLETE PCR KIT DNA Polymerase / 10X PCR Buffer / / mm w/ AB-1301/A/N AB-1301/B/N Red Hot Taq Version (Red Dye w/enzyme) AB-0406/A/N AB-0406/B/N w/ AB-1406/A/N AB-1406/B/N 1.1X Concentration AB-0575/A AB-0575/B 2X Concentration AB-0575/DC/A AB-0575/DC/B MASTER MIX Master Mix containing DNA Polymerase, and Red Dye in enzyme mix helps helps verify enzyme addition during reaction set-up AB-0790/A AB-0790/B ENZYME KIT WITH REDDYMIX DNA Polymerase / 10X ReddyMix PCR Buffer / 400 x 25 μl 4000 x 25 μl w/ AB-0785/A 400 x 25 μl AB-0785/B 4000 x 25 μl Available Pre-Aliquoted into ABgene AB-0790/A/N AB-0790/B/N COMPLETE PCR KIT WITH REDDYMIX DNA Polymerase / 10X ReddyMix PCR Buffer / / mm w/ AB-0785/A/N AB-0785/B/N 1.1X Concentration AB-0575/LD/A AB-0575/LD/B 2X Concentration AB-0575/DC/LD/A AB-0575/DC/LD/B REDDYMIX MASTER MIX ReddyMix Master Mix containing DNA Polymerase, and Notes Save time with ReddyMix! Go straight from PCR to gel loading with ReddyMix Formats Notes 6 Concentrations: All buffers pre-mixed with will be at 1.5 mm concentration in the final reaction. Alternative pre-mixed buffers are available containing concentrations of 2.0, 2.5, 3.0, 3.5, 4.0 mm. Please enquire. KCl Buffer Options: All mixes contain (NH 4 ) 2 SO 4 buffer. KCl based buffers are available upon request. Master Mix Concentrations: 2X Concentration allows for the addition of more template volume to accommodate dilute or low copy number templates (1.1X allows for of up to 1.5 ul of template, 2X allows for up to 11.5 ul of template). Detailed Product Information For more information or to download the product manual, please visit: com/thermoprime

6 Hot-Start PCR Thermo-Start Taq DNA Polymerase Figure 4: Thermo-Start Taq DNA Polymerase remains completely inactive until the 95 o C activation step. Figure 5: Thermo-Start Taq DNA Polymerase exhibits enhanced specificity and sensitivity over other hot-start enzymes. During PCR set-up, reaction samples may be exposed to sub-optimal annealing temperatures, allowing primers to anneal to each other or to non-specific segments of the template. During this time, active DNA polymerase can extend these misaligned primers to create primer-dimers or non-specific product. This amplification of non-specific product continues throughout PCR cycling, consuming reaction components and reducing the amplification efficiency for the target sequence, resulting in reduced assay sensitivity and end yield of the target product. Thermo-Start Taq DNA Polymerase eliminates this non-specific amplification by preventing DNA polymerase activity from occurring during assay set-up. The polymerase activity is suspended using a covalent chemical modification that inhibits all enzyme activity. This modification is maintained until reversed by a 15 minute 95 o C activation step. In addition, the Thermo-Start buffering system contains heat stabilizing components that protect Thermo-Start over the multiple high temperature steps of PCR cycling, to deliver highly efficient, specific amplification of even very low copy number targets. A high performance buffer is also available for GC-rich targets or other challenging templates with a high degree of secondary structure. Hot-Start activation for stringent specificity Hot-Start modification allows for room temperature set-up Enhanced enzyme stability for increased yield High Performance Buffer for GC-rich templates PCR samples are subject to sub-optimal annealing conditions during PCR setup and transport, as well as during the initial denaturation cycle. It is important that the DNA polymerase remains inactive until conditions are optimal for specific amplification. Temperature (ºC) Thermo-Start activation Initial Denaturation Potential for Non-specific amplification Primer Annealing Primer Extension PCR Protocol Steps Cycling Denaturation Sub-optimal Annealing Temperature PCR amplification of a 250 bp and 630 bp β-actin gene fragment from 3 concentrations (100, 50 and 25 ng) of human genomic DNA using either Thermo-Start Taq or an equivalent hot-start Taq from Supplier A, Q or I. Thermo-Start Supplier A Supplier Q Supplier I Genomic DNA Template (ng) >> bp >> 250 bp >> THERMO-START Choose Your Preferred Format: AB-0908/A AB-0908/B ENZYME KIT DNA Polymerase / 10X PCR Buffer / w/ AB-1908/A AB-1908/B w/ High Performance Buffer Formulation AB-1057/A AB-1057/B AB-0908/A/N AB-0908/B/N COMPLETE PCR KIT DNA Polymerase / 10X PCR Buffer / / mm w/ AB-1908/A/N AB-1908/B/N w/ High Performance Buffer Formulation AB-1057/A/N AB-1057/B/N 1.1X Concentration AB-0938/15/A AB-0938/15/B 2X Concentration AB-0938/15/DC/A AB-0938/15/DC/B MASTER MIX Master Mix containing DNA Polymerase, and w/ High Performance Buffer Formulation 2X Concentration AB-1147/A AB-1147/B AB-1950/A AB-1950/B ENZYME KIT WITH REDDYMIX DNA Polymerase / 10X ReddyMix PCR Buffer / w/ High Performance Buffer Formulation AB-1951/A AB-1951/B AB-1950/A/N AB-1950/B/N COMPLETE PCR KIT WITH REDDYMIX High Performance Buffer is recommended exclusively for GC-rich or other complex targets DNA Polymerase / 10X ReddyMix PCR Buffer / / mm w/ High Performance Buffer Formulation AB-1951/A/N AB-1951/B/N 1.1X Concentration AB-1960/A AB-1960/B 2X Concentration AB-1961/A AB-1961/B REDDYMIX MASTER MIX ReddyMix Master Mix containing DNA Polymerase, and w/ High Performance Buffer Formulation 2X Concentration AB-1962/A AB-1962/B Save time with ReddyMix! Go straight from PCR to gel loading with ReddyMix Formats Notes 6 Concentrations: All buffers pre-mixed with will be at 1.5 mm concentration in the final reaction. Alternative pre-mixed buffers are available containing concentrations of 2.0, 2.5, 3.0, 3.5, 4.0 mm. Please enquire. Master Mix Concentrations: 2X Concentration allows for the addition of more template to accommodate dilute or low copy number templates. (1.1X allows for of up to 1.5 ul of template, 2X allows for up to 11.5 ul of template.) Detailed Product Information For more information or to download the product manual, please visit

7 High Fidelity PCR Verbatim High Fidelity DNA Polymerase Figure 7: Verbatim s high affinity to DNA templates increases processivity to deliver improved yield over standard Taq and Pfu. Figure 8: Shortened extension times allow for up to 75% reduction in overall protocol times. Demanding applications, such as cloning and mutagenesis, require a high performance proofreading enzyme that delivers maximum accuracy and yield. Standard proofreading enzymes such as Pfu or KOD have only moderate fidelity and exhibit reduced processivity due to their proofreading activity. This results in a low yield PCR product that contains an increased number of sequence errors. Thermo Scientific Verbatim High-Fidelity DNA Polymerase is an advanced proofreading enzyme that delivers superior fidelity and processivity. The Verbatim enzyme is a modified DNA polymerase with enhanced proofreading activity and high template binding affinity. The enhanced proofreading activity generates highly accurate PCR products, and the increased affinity dramatically improves processivity and yield. Additionally, the increased processivity allows for up to 75% reduction in PCR protocol times for faster results. The high template binding affinity also helps Verbatim work through difficult templates, making it a robust enzyme successful on a wide variety of targets. Verbatim High-Fidelity DNA Polymerase is provided with two optimized buffering systems, our high-fidelity buffer for success with most templates, and a GC buffer for success with GC-rich and other difficult to amplify templates. Superior proofreading capabilities for industry leading fidelity Enhanced processivity for increased yield and up to 75% reduction in protocol times Flexible buffering systems for success with a variety of templatesi PCR amplification of pkf3 plasmid (2.3 kb) using either standard Taq, a Pfu enzyme, or Verbatim with extension times of either 15 sec, 30 sec or 60 sec, during a 40 cycle protocol. All products are run with a 1 kb ladder (AB-0387). 2.8 kb >> kb 15 sec/ kb Taq Ver Pfu 30 sec/ kb 60 sec/ kb Taq Ver Pfu Taq Ver Pfu Calculated PCR amplification protocol times using Pfu, Taq or Verbatim with 1, 2.8, and 5 kb templates. Protocol times are based on suppliers recommended 3-step reaction protocol repeated over 30 cycles. Template Length 1kb 2.8kb 5kb 35 min 1 hour 1 hour Not recommended Total Protocol Time 2 hours 2 hours 3 hours, 45 min PFU Taq Verbatim 6 hours 1 hour, 35 min Figure 6: Verbatim High Fidelity DNA Polymerase exhibits superior fidelity compared to Taq, standard proofreaders, and advanced fusion proofreading enzymes. Average error rates of five PCR enzymes, including Verbatim High Fidelity DNA Polymerase (data generated following a modified protocol based on the rpsl method described by Fuji et al, 1999). (1/ Average Error Rate) 1.60E E+05 1.E E E E E+04 Enzyme Fidelity Choose Your Preferred Format: AB-19/A AB-19/B ENZYME KIT 1 2 DNA Polymerase / 5X High Fidelity Buffer / 5X GC Buffer / 0 x 25 µl rxns 1000 x 25 µl rxns AB-19/A/N AB-19/B/N COMPLETE PCR KIT 1 2 DNA Polymerase / 5X High Fidelity Buffer / 5X GC Buffer / / 0 x 25 µl rxns 1000 x 25µl rxns Verbatim Notes Concentrations: Both the High Fidelity and GC buffers are pre-mixed with at 2.0 mm concentration in the final reaction. The separate vial can be used for further optimization. High Fidelity Buffer and GC Buffer: Use High Fidelity Buffer for all standard templates. Use GC Buffer only for GC rich or other complex templates. T/A Cloning: Due to its proof-reading activity, Verbatim removes the 3 -A overhangs necessary for T/A cloning applications. Protocols are readily available for the addition of 3 -A overhangs post-amplification. Please contact our technical support team for more information. Detailed Product Information For more information or to download the product manual, please visit E E+00 Taq Pfu/Taq Blend KOD Leading Fusion Enzyme Verbatim Available Pre-Aliquoted into ABgene

8 Long Range PCR Extensor Long Range Enzyme Blend Figure 9: The two enzymes in the Extensor blend act synergistically to generate long length PCR products. Figure 10: The Extensor enzyme blend delivers robust amplification of long range targets. When working with longer length targets, it is important to use an appropriate enzyme system to ensure the generation of full length product. Standard Taq DNA polymerase incorporates approximately 1 error in every 9 kb. This error can cause the polymerase reaction to stop, or proceed very slowly, leading to incomplete amplicons. For long PCR, a system composed of a DNA polymerase and a proofreading enzyme, will give successful amplification; the DNA polymerase performs the nucleotide additions, and the proofreading enzyme corrects any mismatched bases, allowing the polymerase to amplify long sections of the template. Extensor Long Range Enzyme Blend is an optimized mix of ThermoPrime DNA Polymerase and a proprietary thermostable proofreading enzyme. The two enzymes act synergistically to generate long PCR products, up to kb. This makes the enzyme blend ideal for robust full-length amplification of long range sequences. PCR products generated with Extensor can be used directly in T/A cloning. Despite the 3 to 5 exonuclease (proofreading) activity present in the mix, the ThermoPrime Taq DNA Polymerase generates sufficient product with an A overhang, eliminating the need to add A overhangs post PCR. Extensor Long Range Enzyme Blend is available with 2 buffer formulations; Buffer 1 for success with most templates up to 12 kb in length, and Buffer 2 for templates 12 - kb and GC-rich or other difficult templates. Proprietary enzyme blend amplifies targets up to kb in length Unique blend and optimized buffer combination delivers increased accuracy and yield Alternative buffering system for success with GC-rich targets PCR amplification of 2, 5, 7, 9, 11, 13 and 15 kb fragments of the human tissue plasminogen activator gene from human genomic DNA and a kb fragment from lambda DNA. Genomic DNA Target ( kb) >> EXTENSOR Choose Your Preferred Format: AB-07/A AB-07/B ENZYME KIT 1 2 Polymerase Blend / 10X PCR Buffer 1 / 10X PCR Buffer 2 / 80 x 25 μl rxns AB-07/A/N AB-07/B/N COMPLETE PCR KIT 1 2 Polymerase Blend / 10X Buffer 1 / 10X Buffer 2 / / mm 80 x 25 μl rxns MASTER MIX Master Mix containing Polymerase Blend, and w/ Buffer 1 Formulation (2X Conc.) AB-0792/A 80 x 25 µl rxns AB-0792/B 400 x 25 µl rxns REDDYMIX MASTER MIX ReddyMix Master Mix containing Polymerase Blend, and w/ Buffer 1 Formulation (2X Conc.) AB-0794/A 80 x 25 µl rxns AB-0794/B 400 x 25 µl rxns Save time with ReddyMix! Go straight from PCR to gel loading with ReddyMix Formats 6 Notes Control Template & Primer Formats: These PCR kits include control genomic DNA with two sets of primers designed to create 5 kb and 15 kb fragments. Intended for use as a positive control in long range PCR. Buffer 1 and Buffer 2: Use Buffer 1 for target sequences up to 12 kb. Use Buffer 2 for targets 12 - kb, GC-rich or problematic targets of any length. Concentrations: All buffers pre-mixed with will be at 1.5 mm concentration in the final reaction. Master Mix Concentrations: 2X Concentration allows for the addition of more template volume to accommodate dilute or low copy number templates. Detailed Product Information: For more information or to download the product manual, please visit w/ Control Template & Primers AB-0721/A 80 x 25 μl rxns AB-0721/B w/ Control Template & Primers AB-0721/A/N 80 x 25 μl rxns AB-0721/B/N w/ Buffer 2 Formulation (2X Conc.) AB-0793/A 80 x 25 µl rxns AB-0793/B 400 x 25 µl rxns w/ Buffer 2 Formulation (2X Conc.) AB-0795/A 80 x 25 µl rxns AB-0795/B 400 x 25 µl rxns Control Template & Primer Formats include genomic DNA with forward and reverse primers Available Pre-Aliquoted into ABgene

9 RT-PCR Verso Reverse Transcriptase Figure 12: Verso exhibits high affinity for mrna templates giving long and high-yield cdna providing an accurate cdna pool for PCR. Figure 13: RT Enhancer degrades genomic DNA, eliminating the need for separate DNase treatment. Successful RT-PCR analysis depends greatly on a robust and sensitive reverse transcription reaction; one with a processive RT enzyme that can effectively transcribe through difficult RNA secondary structure, and a priming method that ensures high cdna yield while maintaining an unbiased representation of the RNA population. The Verso system achieves robust and sensitive reverse transcription through the combination of a high affinity RT enzyme, a unique RNA priming method, and an optimized buffering system. The Verso RT enzyme has high RNA template affinity and reduced RNase H activity, allowing the enzyme to generate long cdna fragments. The generation of long, uninterrupted fragments demonstrates that even sections with high secondary structure are successfully reverse transcribed, ensuring that all RNA sequences are represented in the cdna pool (Figure 12). The included anchored oligo dt priming method further enhances sensitivity by increasing transcription efficiency (Figure 11). There are two buffering options for the Verso enzyme; a cdna synthesis buffer, and a 1-Step RT-PCR buffer. The cdna synthesis buffer has been optimized to achieve a full and diverse cdna pool, and the 1-Step system has been developed to attain high performance from both the reverse transcriptase and the DNA polymerase, delivering superior sensitivity and yield. High affinity RT enzyme for robust cdna synthesis Efficient RT reaction allows for 75% shorter protocol times (down to 30 minutes) Unique priming strategy for broad template success Wide working temperature range for GC rich targets RT Enhancer eliminates the need for DNase treatment Following reverse transcription using the Verso cdna Synthesis Kit, PCR primers pairs were used to amplify short fragments 0.56 kb, 9.7 kb and 11.1 kb from the mrna / poly(a) junction. The successful amplification of all 3 amplicons, even from very low input concentrations, demonstrates that Verso RT has a high affinity for RNA and can reverse transcribe long RNA templates. NTC no template control; NEC no enzyme control. cdna length 0.56 kb 9.7 kb 11.1 kb M 100ng 10ng 1ng 100pg NTC NEC The RT Enhancer, which is included in all Verso kits, is a unique enzyme that degrades contaminating genomic DNA at the start of the RT step. It is equally as effective as DNase I treatment but more convenient as it eliminates the need for a separate DNase I incubation prior to RT-PCR. Figure below demonstrates equal DNA degradation with RT enhancer and DNase I treatment. M M Genomic DNA treated with: 1 (No treatment) 2 RT Enhancer 3 (No treatment) 4 DNase treatment M = DNA Marker Figure 11: Utilizing Anchored oligo dt in place of standard oligo dt increases reverse transcription efficiency. Standard oligo dt anneals anywhere along the poly A tail, and can therefore prime internal sites within the poly A tail or other poly A rich regions. Standard oligo dt 3 5 TTTTTTTTTT AAAAAAAAAAAAAAA mrna 5 3 Anchored oligo dt anneals only at the mrna / poly A tail junction, preventing inefficient inner A tail priming and increasing mrna transcription efficiency. Anchored oligo dt 3 5 N V TTTTTTTTTT AAAAAAAAAAAAAAA mrna 5 3 V dgtp, datp or dctp any base VERSO RT PCR AB-1453/A AB-1453/B 2-Step RT-PCR: cdna SYNTHESIS KIT RT ENZYME PRIMER 1 PRIMER 2 RT Enzyme / 5X RT Buffer / Random Hexamers / Anchored Oligo dt / mm / RT Enhancer 40 x μl rxns 100 x μl rxns Pair with one of our PCR kits for complete 2-Step RT-PCR PCR Kit Standard PCR 7 High Yield PCR 8 Hot-Start PCR 10 High Fidelity PCR 12 Long Range PCR 14 RT ENH. 1-Step RT-PCR: 1-STEP RT-PCR MASTER MIX RT ENZYME RT ENH. RT Enzyme / 2X Master Mix Buffer containing DNA Polymerase, and / RT Enhancer w/ ThermoPrime Taq DNA Polymerase AB-1454/A 80 x 25 µl rxns AB-1454/B 400 x 25 µl rxns Hot-Start Version w/ Thermo-Start Taq DNA Polymerase AB-1455/A 80 x 25 µl rxns AB-1455/B 400 x 25 µl rxns 1-STEP RT-PCR REDDYMIX MASTER MIX RT ENZYME RT ENH. RT Enzyme / 2X Master Mix ReddyMix Buffer containing DNA Polymerase, and / RT Enhancer w/ ThermoPrime Taq DNA Polymerase AB-1454/LD/A 80 x 25 μl rxns AB-1454/LD/B Available Pre-Aliquoted into ABgene Save time with ReddyMix! Go straight from PCR to gel loading with ReddyMix Formats 6 RT Controls: AB-1456/A RNA Control Kit template PRIMER 1 Positive Control RNA / Control Primer 1 / Control Primer 2 40 x 25 μl rxns RNA Control Kit The RNA control kit is intended for use as a positive control to monitor the performance of all RT PCR components. The kit includes a 3.6 kb RNA synthesized according to the sequence derived from bacteriophage MS2 and primers designed to generate a 522 bp product. PRIMER

10 ABgene PCR Tubes, Plates and Seals High quality PCR plastics are an important factor in achieving sensitive and accurate PCR results. The wells must deliver fast, uniform heat transfer for maxi mum and consistent PCR efficiency. Plates and seals must be engineered to work together to prote ct samples from evaporation, and the plastics must be manufactured in a process that avoids impurities and contaminants that can affect molecular biology applications. Thermo Scientific ABgene PCR plastics are cleanroom produced and expertly manufactured to ensure maximum PCR performance. To create a highly inert well surface, all plastics are made from virgin medical grade polypropylene, and molding tools are maintained without the use of lubricating agents. Molds are precision designed and highly polished to create uniform, ultra thin, smooth well walls. The quick order tables below represent some of the most popular formats, but the full range of formats and product options is available online and in our PCR plastics selection guide. (See below for details) Cleanroom produced for contamination-free PCR Uniform, ultra-thin well walls for maximum efficiency and reproducibility Plates and seals are precision designed to minimize evaporation RNase, DNase, Endotoxin, and Human DNA free RNase DNase Endotoxins Human DNA Plates To view our complete range of compatible plates, please refer to our full guide. Popular Formats: Clear AB-0600 Assorted Colors AB-0600/B AB-0600/G AB-0600/P AB-0600/R AB-0600/Y 96-WELL PCR PLATE, NON-SKIRTED Clear AB-1400 Clear with Barcodes BC WELL PCR PLATE, SEMI-SKIRTED Compatible with most 0.2 ml 96-Well cycler blocks. Clear AB-2150 Flat Caps BC WELL DIAMOND ULTRA PCR PLATE 50 Plates 50 Plates Compatible with most 384-Well cycler blocks. Compatible with most 0.2 ml 96-Well cycler blocks. For a full list of compatible instruments, visit Tubes Popular Formats: 0.2 ML DOMED CAP PCR TUBES 0.2 ML FLAT CAP PCR TUBES 0.2 ML EASYSTRIP PCR TUBES Seals Popular Formats: ADHESIVE PCR FOIL SEAL ADHESIVE PCR FILM SEAL 8-STRIP PCR CAP STRIPS More options available, please refer to our full PCR Plastics guide. Clear AB Tubes Assorted Colors AB Tubes Compatible with most 0.2 ml cycler blocks Clear AB Tubes Assorted Colors AB Tubes Compatible with most 0.2 ml cycler blocks Domed Caps (individually attached) AB Strips Flat Caps (individually attached) AB Strips Compatible with most 0.2 ml cycler blocks For more sealing options, please refer to our full PCR Plastics guide. AB Seals AB Seals Domed AB-0265 Flat AB Strips 250 Strips To view the complete range of Thermo Scientific ABgene plastics or to order our Selection Guide, please visit:

11 Custom Aliquoting Services In-House HTP Assay Set-up and Management VS Thermo Scientific Custom Aliquoting Services Custom Solutions for High-Throughput PCR Thermo Scientific Custom Aliquoting Services can significantly streamline your process and increase your PCR productivity. Our Custom Aliquoting Service can provide any of our PCR and qpcr mixes pre-aliquoted into our high quality PCR plastics. Save time, money, and effort by letting our PCR focused manufacturing facility, state-of-the-art instruments, and skilled production staff supply you with reliable, quality controlled, pre-filled PCR plastics. PCR Plastic Format Options Thermo Scientific ABgene PCR plastics are expertly designed and cleanroom produced for the ultimate in PCR reliability. All products are manufactured under strict ISO 9001 guidelines and go through extensive QC testing to ensure PCR performance. Our quality PCR plastics are available in a variety of formats to fit any cycler and suit any workflow. INDIVIDUAL TUBES STRIP TUBES Multiple Suppliers For every separate product that is purchased, there are separate orders, invoices, and stocking. Procurement and management of these components is a significant investment of time and resources. Multiple QC/QA Documents Each separate component has its own QC documentation, and multiple lot numbers for each component add to the complexity of QC management. In-house Production of Master Mix 96-WELL PLATES 384-WELL PLATES Multiple In-house Set-up Steps Having multiple in-house set-up steps introduces variables into your production process. This increases the likelihood of failure, and makes troubleshooting a long and difficult process. 04 Improve Quality Control and Documentation Buying pre-filled PCR plastics greatly simplifies the procurement process. All reagent and plastic components are ordered with a single part number and can be purchased in a single transaction. Standing orders can be set up to deliver regular shipments to match production demand. The supply chain is simplified by having one vendor for all PCR consumables, and stock management is far easier when the products arrive in a pre-assembled format. Stop collecting and managing QC documentation from multiple suppliers, for multiple products and lot numbers. All of our pre-aliquoted products are manufactured under strict ISO 9000 quality control guidelines and are supplied with certificates of analysis for each reagent and plastics lot. We can also provide entire orders consisting of a single combination of plastic and reagent lot numbers for easy QC tracking. End the struggle to manage QC data and increase assay reproducibility by switching to our pre-filled PCR plastics. Reduce Assay Failure and Increase Productivity Increase productivity by letting us take care of your reaction mix and aliquoting steps. In-house master mix creation and aliquoting can introduce multiple variables in your production process. Don t waste valuable time and resources troubleshooting through multiple production steps. Let our 24/7 PCR manufacturing facility provide you with an uninterrupted supply of cycler-ready, reliable product to keep your project moving. Minimal In-house Set-up Steps Most in-house set-up steps are eliminated with pre-filled plastics. Our PCR focused manufacturing facility operates under ISO 9001 guidelines to create, assemble and deliver production quality pre-filled plates. Add Template and Primers Let Thermo Scientific Custom Aliquoting Services Streamline Your PCR Workflow Simplify Supply Chain & Materials Management Simplified QC/QA Documentation Produced under strict QC guidelines and can be supplied with QC documentation for reagent and plastic. Orders can be made up of single lot numbers for reagent and plastic. Single Supplier Plastics and reagents are all from the same supplier, simplifying ordering and materials maintenance and storage. Run PCR & Analyze results Dispense Master Mix To learn more about our custom aliquoting solutions, please visit: Add Template and Primers Run PCR & Analyze results 21

12 Ordering Information United States Fisher Scientific: (All Products) Direct: (PCR Reagents Only) Telephone: (800) Telephone (800) To fax an order: (800) To fax an order (800) Order online: Product Information: United Kingdom Direct: (All Products) Telephone: To fax an order: Order online: France Direct: (PCR Reagents Only) Dominique Dutscher: (All Products) Fisher Scientific: (All Products) Telephone: Telephone: Telephone: To fax an order: To fax an order: To fax an order: Order online: Order online: Order online: Germany Direct: (All Products) Telephone: To fax an order: Order online: Rest of World Please refer to our distributor listing by visiting Need help? Contact your local technical support team United States Telephone: (800) , option #4 United Kingdom Telephone: France Telephone: Germany Telephone: Rest of World Telephone:

13 Thermo Scientific PCR Reagent Selection Guide 09 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries