[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[qPCR](https://www.sigmaaldrich.com/US/en/applications/genomics/qpcr)Primer Concentration Optimization Protocol # Primer Concentration Optimization Protocol ## Optimization of qPCR Conditions Optimization of qPCR conditions is important for the development of a robust assay. 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 optimizing primer concentrations is to create a matrix of reactions. This is used to test a range of concentrations for each primer against different concentrations of the partner primer. In the example provided in this protocol, a 6×6 matrix testing six concentrations (e.g., 50 nM to 800 nM) is demonstrated. The quantities stated in this protocol will allow each condition to be run in duplicate. ## Equipment used in Primer Concentration Optimization Protocol - Quantitative PCR instrument - Laminar flow hood for PCR set up (optional) ## Reagents - Suitable assay template, e.g., cDNA diluted 1:10, gDNA, or synthetic oligo template. - KiCqStart® SYBR® Green ReadyMix™ (KCQS00/KCQS01/KCQS02/KCQS03—depending on instrument, see __Table P4-6__ for instrument compatibility). - PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction. - Forward and reverse primers concentration stocks (10 μM working stocks). •    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)KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox (Cat. No. KCQS01)KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX (Cat. No. KCQS02)KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ (Cat. No. 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 P13-31.SYBR Green PCR Mix Selection Guide. ## Supplies - Sterile filter [pipette tips](https://www.sigmaaldrich.com/US/en/products/labware/liquid-handling-and-dispensing/pipette-tips) - Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909) - PCR tubes and plates, select one to match desired format: • Individual thin-walled 200 μL PCR tubes (Z374873 or P3114) • Plates   - 96-well plates (Z374903)   - 384-well plates (Z374911) • Plate seals   - ThermalSeal RTS™ Sealing Films (Z734438)   - ThermalSeal RT2RR™ Film (Z722553) ### Notes for this Protocol - cDNA is generated using a random primer or oligo-dT priming method and diluted 1:10 for use, but any suitable, alternative template may be used. - All samples are run in duplicate according to the plate layout (__Figure P13-18__).  __Figure P13-18.__Schematic Representation of the Primer Optimization Plate Layout. ## Method *__Note:__* 2.0 μL of each primer will be added to the reaction of 20 μL total volume. For this reason, primer stocks are 10 times the required concentration to achieve the desired final concentration. 1.    Using the 10 μM primer stock, make a dilution of both primer stocks to 0.5, 1, 2, 4, 6 and 8 μM as shown in        __Table P13-32__. Final Concentration (μM)Volume (μL) H2OVolume (μL) 10 μM StockTotal Volume (μL) 0.547.52.550 145550 2401050 4302050 6203050 82080100 Table P13-32.Primer Dilution Scheme for Primer Concentration Optimization. 2.    Prepare a qPCR master mix according to __Table P13-33__           (*__Note:__* Template and cDNA are added separately in step 5). Mix well. ReagentsVolume (μL) per Single 20 μL ReactionVolume (μL) for Whole Plate (84+10% pipetting error)   2× KiCqStart® SYBR® Green qPCR ReadyMix™10924 PCR grade water3277.2 Reference dye (Optional)192.4 Template, e.g., cDNA (diluted 1:5 to 1:10 of stock)2* Forward primer2* Reverse primer2* Table P13-33.Reaction Master Mix for Primer Concentration Optimization. *__\*Note:__* Do __not__ add cDNA and primers until step 5. 3.    Remove 184.8 μL (for 12× NTC) of master mix from step 2 into a separate tube to use for setting up the        No Template Control (NTC). 4.    Add 26.4 μL of dH2O to the NTC mix in step 3 (to replace template).        *Note:* Set NTC mix on ice for later use. 5.    Add 158.4 μL of cDNA template to the remaining master mix from step 2. Set master mix on ice. 6.    Add 2.0 μL of appropriate reverse primer dilutions into the PCR plate according to __Figure P13-18__; also adding        800 nM concentration to the NTC row. 7.    Add 2.0 μL of appropriate forward primer dilutions into the PCR plate according to __Figure P13-18__. 8.    Aliquot 16 μL master mix from step 5 into the PCR plate in the wells corresponding to test positions. 9.    Aliquot 16 μL master mix from step 4 into the PCR plate for NTC reactions. 10.  Seal plates and label. (Make sure labeling does not obscure instrument excitation/detection light path). 11.  Run samples according to the two-step protocol in __Table P13-34__. Steps 1 and 2 are repeated through 40 cycles and followed by a dissociation curve analysis. | | | | |----------------------------------------------|-----------|------------| | Cycling Conditions | Temp (°C) | Time (sec) | | Initial denaturation/Hot Start | 95 | 30 | | __Steps 1–2 are repeated through 40 Cycles__ | | | | Step 1 | 95 | 5 | | Step 2 | 60 | 30 | Table P13-34.PCR Cycling Conditions for Primer Concentration Optimization. *__Note:__* Use standard dissociation curve protocol (data collection). ## Materials Sorry, an unexpected error has occurred Response not successful: Received status code 500 __Related Articles__ - [PCR/qPCR/dPCR Assay Design](https://www.sigmaaldrich.com/US/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/US/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/US/en/technical-documents/technical-article/genomics/dna-and-rna-purification/pcr-assay-development) - [Water for Next-Generation Sequencing](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/next-gen-sequencing/water-for-next-generation-sequencing) - [PCR Assay Optimization and Validation](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/assay-optimization-and-validation) - [DNA Oligonucleotide Synthesis](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis) - [Locked Nucleic Acid & Minor Groove Binder / Eclipse Dark Quencher](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/locked-nucleic-acids-faq) - [PCR Technologies Guide](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/pcr-technologies-protocols-introduction) - [View More](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) __Related Product Categories__ - [Sealing Films, Foils & Tapes](https://www.sigmaaldrich.com/US/en/products/labware/sealing-films-foils-and-tapes) - [Pipette Tips](https://www.sigmaaldrich.com/US/en/products/labware/liquid-handling-and-dispensing/pipette-tips) Top __Sign In To Continue__ To continue reading please sign in or create an account. 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