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Indications of poor optimization are a lack of reproducibility between replicates as well as inefficient and insensitive assays. The two main approaches are optimization of primer concentration and/or annealing temperatures.  One approach to assay optimization is to determine the optimum annealing temperature (Ta) of the primers by testing identical reactions containing a fixed primer concentration, across a range of annealing temperatures. This can be achieved if a qPCR instrument with a temperature gradient block is available. In the format presented in this protocol, primers are included at a final concentration of 450 nM, and the gradient is orientated across the X axis of the block such that all columns are subjected to the same Ta (i.e., 12 different temperatures). In other instruments, the gradient is down the column such that all rows have the same Ta (i.e., 8 different temperatures). The following protocol can be applied to either, albeit after minor modifications. ## Equipment - Quantitative PCR instrument with integrated gradient block control function - Laminar flow hood for PCR set up (optional) ## Reagents - cDNA diluted 1:5-1:10, gDNA 10ng, synthetic oligo (10,000 copies) or other suitable template for optimization. - KiCqStart SYBR® Green ReadyMix™ ([KCQS00/KCQS01/KCQS02/KCQS03](https://www.sigmaaldrich.com/BR/en/product/sigma/KCQS00); instrument specific, see Table P17-42). - PCR grade water: PCR grade water ([W1754](https://www.sigmaaldrich.com/BR/en/product/sigma/W1754) or [W4502](https://www.sigmaaldrich.com/BR/en/product/sigma/W4502)) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction. - Forward and reverse primers concentrated stocks (10 μM working stocks: GOI). •    Custom oligos can be ordered at sigmaaldrich.com/oligos. Hot Start ReadyMixes (Taq, Buffer, dNTPs, Reference Dye, MgCl2) KiCqStart® SYBR® Green qPCR ReadyMix™, [Cat. No. KCQS00](https://www.sigmaaldrich.com/BR/en/product/sigma/KCQS00)KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox , [Cat. No. KCQS01](https://www.sigmaaldrich.com/BR/en/product/sigma/KCQS01)KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX, [Cat. No. KCQS02](https://www.sigmaaldrich.com/BR/en/product/sigma/KCQS02)KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ, [Cat. No. KCQS03](https://www.sigmaaldrich.com/BR/en/product/sigma/KCQS03) __Compatible Instruments:____Compatible Instruments:____Compatible Instruments:____Compatible Instruments:__ Bio-Rad CFX384™Applied Biosystems 7500Applied Biosystems 5700Bio-Rad iCycler iQ™ Bio-Rad CFX96™Applied Biosystems 7500Applied Biosystems 7000Bio-Rad iQ™5 Bio-Rad MiniOpticon™Fast Applied Biosystems ViiA 7Applied Biosystems 7300Bio-Rad MyiQ™ Bio-Rad MyiQ™Stratagene Mx3000P®Applied Biosystems 7700  Bio-Rad/MJ Chromo4™Stratagene Mx3005P™Applied Biosystems 7900  Bio-Rad/MJ Opticon 2Stratagene Mx4000™Applied Biosystems 7900 HT Fast  Bio-Rad/MJ Opticon® Applied Biosystems 7900HT  Cepheid SmartCycler® Applied Biosystems StepOnePlus™  Eppendorf Mastercycler® ep realplex Applied Biosystems StepOne™  Eppendorf Mastercycler® ep realplex2 s    Illumina Eco qPCR    Qiagen/Corbett Rotor-Gene® 3000    Qiagen/Corbett Rotor-Gene® 6000    Qiagen/Corbett Rotor-Gene® Q    Roche LightCycler® 480    Table P17-42. SYBR Green PCR Mix Selection Guide ## Supplies - Sterile filter [pipette tips](https://www.sigmaaldrich.com/BR/en/products/labware/liquid-handling-and-dispensing/pipette-tips) - [Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)](https://www.sigmaaldrich.com/BR/en/product/sigma/CLS430909) - PCR tubes and plates, select one to match desired format: •    Individual thin-walled 200 μL PCR tubes ([Z374873](https://www.sigmaaldrich.com/BR/en/product/sigma/Z374873) or [P3114](https://www.sigmaaldrich.com/BR/en/product/sigma/P3114)) •    Plates       - [96-well plates (Z374903)](https://www.sigmaaldrich.com/BR/en/product/sigma/Z374903)       - [384-well plates (Z374911)](https://www.sigmaaldrich.com/BR/en/product/sigma/Z374911) •    Plate seals       - [ThermalSeal RTS™ Sealing Films (Z734438)](https://www.sigmaaldrich.com/BR/en/product/sigma/Z734438)       - [ThermalSeal RT2RR™ Film  (Z722553)](https://www.sigmaaldrich.com/BR/en/product/sigma/Z722553) ### Notes for this Protocol - cDNA is generated using random priming or oligo-dT priming method and diluted 1:10 for use, but any suitable, alternative template may be used. - All reactions are run in duplicate as technical replicates. - If using a PCR plate, follow a plate schematic (e.g., shown in __Figure P14-19__) to ensure that the reaction mix, samples and controls are added to the correct wells.   ## Method 1.    Prepare a master mix for 56 reactions according to __Table P14-35__. Mix well, avoiding bubbles. ReagentsVolume (μL) per Single 20 μL ReactionVolume (μL) for 56 Reactions KiCqStart SYBR® Green qPCR ReadyMix 2×10560 Forward primer (10 μM)0.950.4 Reverse primer (10 μM)0.950.4 PCR grade water4.2235.2 Table P14-35. Reaction Master Mix for Ta Optimization 2.    Remove 448 μL of master mix from step 1 (i.e., half ) into a separate tube for setting up the No Template Control (NTC)        reactions. 3.    Add 112 μL of template to the remaining master mix from step 2. Set Template master mix on ice. 4.    Add 112 μL of water to the other half of the master mix from step 2. Set NTC master mix on ice. 5.    Aliquot 20 μL Template master mix from step 3 into two rows of the PCR plate labeled GOI. 6.    Aliquot 20 μL NTC master mix from step 4 into two rows of the PCR plate labeled NTC. 7.    Cover plates and label. (Make sure the labeling does not obscure instrument excitation/detection light path.) 8.    Run samples according to the three-step protocol below        (*__Note:__* These conditions are specific for FAST cycling protocols) ensuring that the annealing temperature has been        defined on a gradient between the lowest and highest that would be appropriate for the primers (example shows        54–70 °C). Steps 1–3 are repeated through 40 cycles. A standard dissociation curve is run after amplification. | | | | |----------------------------------------------|------------------|------------| | FAST Cycling Conditions | Temp (°C) | Time (sec) | | Initial denaturation/Hot Start | 95 | 30 | | __Steps 1–3 are repeated through 40 cycles__ | | | | Step 1 | 95 | 5 | | Step 2 | 54–70 (gradient) | 15 | | Step 3 | 72 | 10 | Table P14-36. FAST qPCR Cycling Conditions for Ta Optimization *__Note:__* Use standard dissociation curve protocol (data collection). ![Plate Layout for Ta Optimization With Identical Ta for Each Column.](https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/images/technical-documents/protocols/genomics/qpcr/the-distribution-of-the-samples/the-distribution-of-the-samples.jpg "Plate Layout for Ta Optimization With Identical Ta for Each Column.") __Figure P14-19.__ Plate Layout for Ta Optimization With Identical Ta for Each Column. *__Note:__* The distribution of the samples and controls across the temperature gradient. If the instrument has a temperature gradient that varies vertically down the plate column, the samples and controls will need to be re-arranged accordingly. ## Materials Desculpe, ocorreu um erro inesperado Response not successful: Received status code 500 __Related Articles__ - [PCR/qPCR/dPCR Assay Design](https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/genomics/pcr/pcr-qpcr-dpcr-assay-design) - [Mgat4 May Play a Role in Increased Sialylation by Overexpressing Functional MGAT1 in Mgat1-Disrupted Chinese Hamster Ovary (CHO) Cells](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/genomics/cloning-and-expression/mgat1-disrupted-chinese-hamster-ovary-cells) - [Complete Solutions for PCR Assay Development](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/genomics/dna-and-rna-purification/pcr-assay-development) - [PCR Assay Optimization and Validation](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/genomics/pcr/assay-optimization-and-validation) - [DNA Oligonucleotide Synthesis](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis) - [Locked Nucleic Acid](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/genomics/pcr/locked-nucleic-acids-faq) - [PCR Technologies Guide](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/genomics/pcr/pcr-technologies-protocols-introduction) - [Oligonucleotide Quantification](https://www.sigmaaldrich.com/BR/en/technical-documents/technical-article/genomics/pcr/quantitation-of-oligos) - [View More](https://www.sigmaaldrich.com/BR/en/search/facet-search?focus=sitecontent&term=facet-search) __Related Product Categories__ - [Sealing Films, Foils & Tapes](https://www.sigmaaldrich.com/BR/en/products/labware/sealing-films-foils-and-tapes) - [Pipette Tips](https://www.sigmaaldrich.com/BR/en/products/labware/liquid-handling-and-dispensing/pipette-tips) Topo __Faça login para continuar__ Para continuar lendo, faça login ou crie uma conta. 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[Filtros de vírus](https://www.sigmaaldrich.com/BR/pt/products/pharma-and-biopharma-manufacturing/viral-clearance/virus-filters) - [Inativação de vírus](https://www.sigmaaldrich.com/BR/pt/products/pharma-and-biopharma-manufacturing/viral-clearance/virus-inactivation) - [Biologia de proteínas](https://www.sigmaaldrich.com/BR/pt/products/protein-biology) - [Anticorpos](https://www.sigmaaldrich.com/BR/pt/products/protein-biology/antibodies) - [Anticorpos de validação aprimorados](https://www.sigmaaldrich.com/BR/pt/products/protein-biology/antibodies/enhanced-validation-ab) - [Anticorpos para citometria de fluxo](https://www.sigmaaldrich.com/BR/pt/products/protein-biology/antibodies/flow-cytometry-antibodies) - [Anticorpos Prestige<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/products/protein-biology/antibodies/prestige-antibodies) - [Anticorpos primários](https://www.sigmaaldrich.com/BR/pt/products/protein-biology/antibodies/primary-antibodies) - [Anticorpos secundários](https://www.sigmaaldrich.com/BR/pt/products/protein-biology/antibodies/secondary-antibodies) - 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[Expressão de proteínas](https://www.sigmaaldrich.com/BR/pt/applications/protein-biology/protein-expression) - [Marcação e modificação de proteínas](https://www.sigmaaldrich.com/BR/pt/applications/protein-biology/protein-labeling-and-modification) - [Pulldown de proteína](https://www.sigmaaldrich.com/BR/pt/applications/protein-biology/protein-pulldown) - [Purificação de proteínas](https://www.sigmaaldrich.com/BR/pt/applications/protein-biology/protein-purification) - [Quantificação de proteínas](https://www.sigmaaldrich.com/BR/pt/applications/protein-biology/protein-quantitation) - [Análise estrutural de proteínas](https://www.sigmaaldrich.com/BR/pt/applications/protein-biology/protein-structural-analysis) - [Western Blotting](https://www.sigmaaldrich.com/BR/pt/applications/protein-biology/western-blotting) - [Espectrometria de massa de proteínas](https://www.sigmaaldrich.com/BR/pt/applications/protein-biology/protein-mass-spectrometry) - [Áreas de pesquisa e doenças](https://www.sigmaaldrich.com/BR/pt/applications/research-disease-areas) - 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[Água para química analítica](https://www.sigmaaldrich.com/BR/pt/applications/water-purification/water-analytical-chemistry) - [Água para Biologia Celular e Molecular](https://www.sigmaaldrich.com/BR/pt/applications/water-purification/water-cell-molecular-biology) - [Água para diagnósticos clínicos](https://www.sigmaaldrich.com/BR/pt/applications/water-purification/water-clinical-diagnostics) - [Entendendo a água de laboratório](https://www.sigmaaldrich.com/BR/pt/applications/water-purification/understanding-lab-water) - Serviços - [Fabricação por contrato](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing) - [ADC e bioconjugação](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/adc-bioconjugation) - [Serviços de diagnóstico para OEMs e CMOs](https://www.sigmaaldrich.com/BR/pt/services/ivd-development-manufacturing/diagnostic-oem-cmo) - [mRNA e LNP](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/mrna-and-lnp-formulation-ctdmo-services) - [Serviços de CDMO de lipídios](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/mrna-and-lnp-formulation-ctdmo-services/lipids-cdmo-services) - [Serviço CDMO de nanopartículas de lipídios](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/mrna-and-lnp-formulation-ctdmo-services/lipid-nanoparticle-cdmo-services) - [Molécula pequena](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/small-molecule) - [Polietilenoglicol ativado (PEGs)](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/peg) - [APIs](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/complex-apis) - [APIs altamente potentes](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/high-potent-apis) - [Vetor viral](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/viral-vector-manufacturing) - [Produção de AAV](https://www.sigmaaldrich.com/BR/pt/services/contract-manufacturing/viral-vector-manufacturing/aav-production) - 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[Software de controle e automação de bioprocessamento](https://www.sigmaaldrich.com/BR/pt/integrated-offerings/biopharma-4-0/bioprocessing-automation-control-software) - [Software de análise de dados de bioprocessamento](https://www.sigmaaldrich.com/BR/pt/integrated-offerings/biopharma-4-0/bioprocessing-data-analytics-software) - [Plataforma de triagem Catalexis Catalysts](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/catalexis-catalyst-screening-platform) - [Materiais de Referência Digitais Chemistwin<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/digital-reference-materials) - [DEL Serviço Digital Estendido](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/dna-encoded-library-technology-drug-discovery) - [Ferramenta DOZN](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/dozn-tool) - [Configurador de Meios e Produtos Químicos para Cultura Celular mConfig™](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/mconfig-cell-culture-media-chemicals-configurator) - [Software de registro de teste eletrônico M-Trace<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/m-trace-electronic-test-record-software) - [Millicell<sup>®</sup> Serviços em nuvem](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/millicell-cloud-services) - [MQuant<sup>®</sup> StripScan](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/mquant-stripscan) - [Serviços digitais MyMilli-Q<sup>™</sup>](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/mymilli-q-digital-services) - [SYNTHIA<sup>™</sup> Software de Retrosíntese](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/synthia-retrosynthesis-software) - [Aplicativo Val@M™ para atividades de validação](https://www.sigmaaldrich.com/BR/pt/services/software-and-digital-platforms/valatm-validation-platform) - [Desenvolvimento e fabricação de IVD](https://www.sigmaaldrich.com/BR/pt/services/ivd-development-manufacturing) - [Serviços de desenvolvimento e fabricação de anticorpos para diagnósticos personalizados](https://www.sigmaaldrich.com/BR/pt/services/ivd-development-manufacturing/custom-diagnostic-antibody-development-and-manufacturing-services) - [Serviços de diagnóstico OEM e CMO](https://www.sigmaaldrich.com/BR/pt/services/ivd-development-manufacturing/diagnostic-oem-cmo) - [Serviços de desenvolvimento de ensaios de fluxo lateral](https://www.sigmaaldrich.com/BR/pt/services/ivd-development-manufacturing/lateral-flow-services) - [Serviços de produtos](https://www.sigmaaldrich.com/BR/pt/services/product-services) - [Serviços de aplicação para formulações de doses sólidas](https://www.sigmaaldrich.com/BR/pt/services/product-services/application-services-solid-dose-formulations) - [Calibração e verificação](https://www.sigmaaldrich.com/BR/pt/services/product-services/calibration-and-verification) - [Calibração e verificação do Milli-Q<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/services/product-services/calibration-and-verification/calibration-verification-suitability-testing-of-milli-q-water-pu) - [Sistemas de teste de microbiologia](https://www.sigmaaldrich.com/BR/pt/services/product-services/calibration-and-verification/calibration-verification-microbiology) - [Programa Elevate](https://www.sigmaaldrich.com/BR/pt/services/product-services/elevate-quality-program) - [Programa eMERGE](https://www.sigmaaldrich.com/BR/pt/services/product-services/the-emerge-standard-edata-exchange-platform) - [Programa Emprove<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/services/product-services/emprove-program) - [Meios de cultura celular Emprove<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/services/product-services/emprove-program/emprove-cell-culture-media) - [Portfólio de produtos químicos Emprove<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/services/product-services/emprove-program/emprove-chemicals-portfolio) - [Portfólio de Cromatografia Emprove<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/services/product-services/emprove-program/emprove-chromatography-portfolio) - [Portfólio de filtros e componentes de uso único Emprove<sup>®</sup>](https://www.sigmaaldrich.com/BR/pt/services/product-services/emprove-program/emprove-filter-and-single-use-component-portfolio) - 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