Dye-based qPCR

Product description The 2×miRNA qPCR Fluore Green Master Mix is a new-generation premixed fluorescent quantitative PCR detection reagent specifically developed for miRNA quantification. It contains a special ROX Passive Reference Dye, which is compatible with all qPCR instruments without the need to adjust the ROX concentration for different instruments. The DNA Polymerase used is a chemically modified hot-start polymerase, combined with a special buffer system, which enhances the specificity of the reaction, improves sensitivity, and allows for accurate quantification over a broader range. This product must be used in conjunction with our miRNA First-Strand cDNA Synthesis Reverse Transcription Kit (Poly(A) tailing method, for qPCR) (Cat#N132068) to obtain the final experimental results. Specifications Catalog Number N132039E N132039S Specifications 100 T（20 μL/rxn） 500T（20 μL/rxn） Components Component Identification Component Name N132036E N132036S N132039-A 2×miRNA qPCR Master Mix 1 mL 5×1 mL N132039-B RNase-free H2O 2×1 mL 5×1 mL Storage Ice bag transportation. Store at -20℃ in the dark. Valid for 12 months. Notes 1. Thaw the components completely and gently mix them before use. 2. During the experiment, use contamination-free consumables whenever possible to avoid contamination. 3. Avoid repeated freeze-thaw cycles for this product. Protect from strong light during preparation. 4. For your safety and health, please wear a lab coat and disposable gloves when operating. 5. This product is for research use only. Instructions 1. Reaction Preparation 1) Place the reagents at room temperature to thaw. Before use, invert the reagents gently to mix and then briefly centrifuge to collect any residual liquid from the cap. Avoid vigorous shaking or vortexing to prevent foaming. 2) Keep the reagents on ice and prepare the reaction mixture according to the table below. RNase-free H2O can be substituted with other nuclease-free water used for molecular biology experiments. 【Note】Failure to mix the reagents properly, using a vortex mixer, or not preparing the reagents on ice can lead to decreased reaction performance. Preparation of Quantitative PCR Reaction Mixture Component Volume（μL） Volume（μL） Final concentration 2×miRNA qPCR Master Mix 10 μL 25 μL 1 × Forward Primer（self-provided） X X 400 nM Reverse Primer (10 μM) 0.8 μL 2 μL 400 nM cDNA X X - RNase-free H2O Up to 20 μL Up to 50 μL - 【Note】a. The volume of miRNA first-strand cDNA added should not exceed 1/10 of the total qRT-PCR reaction volume. High concentrations of cDNA may lead to non-specific amplification; it is recommended to dilute the cDNA 10-1000 times as appropriate. b. The Reverse Primer (10 μM) is sourced from Kit N132068. Due to patent confidentiality, the downstream primer sequence for miRNA poly(A) tailing reverse transcription may vary between manufacturers. It is recommended to use Kit N132068 to ensure reliable experimental results. 2.  PCR Reaction Program Setup You can refer to the following two programs for quantitative PCR reactions. 1) Conventional Fluorescent Quantitative PCR Amplification Program (Two-Step Method) Recycling procedure Temperature Time Cycle Number Pre-denaturation 95℃ 10 min 1 Denaturation 95℃ 15 sec 35-40 Annealing/extension 60℃ 20 sec Melting Curve Analysis Default Settings 1 2) Fluorescent Quantitative PCR Fast Amplification Program (Two-Step Method) Recycling procedure Temperature Time Cycle Number Pre-denaturation 95℃ 10 sec 1 Denaturation 95℃ 5 sec 40 Annealing/extension 60℃ 20 sec Melting Curve Analysis Default Settings 1 【Note】The annealing/extension temperature and time can be adjusted according to experimental requirements.The choice between the conventional and fast programs depends on the PCR instrument being used. For example, instruments like the ABI QuantStudio 5 can be set to a fast program, while instruments like the Bio-Rad CFX96 do not have a fast program setting and require the conventional program to be used.  

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Product description The 2×Color qPCR Fluore Green Master Mix (High Rox) is a 2× real-time quantitative PCR amplification premix, characterized by high fluorescence intensity, high sensitivity, strong specificity, and high amplification yield. The core component, Taq DNA polymerase, utilizes an antibody-mediated hot-start mechanism, which effectively inhibits non-specific amplification caused by primer annealing during sample preparation. Additionally, the mix contains factors that enhance PCR amplification efficiency and promote balanced amplification across a wide range of GC content (30-70%), ensuring good linearity in quantitative PCR over a broad quantification range. This product employs a color-changing reaction based on the mixture of different dyes to monitor pipetting operations, effectively reducing the occurrence of pipetting errors. Specifications Catalog Number N132036E N132036S Specifications 50 T（20 μL/rxn） 250 T（20 μL/rxn） Components Component Identification Component Name N132036E N132036S N132036-A 2×Color qPCR Master Mix (High Rox) 1 mL 5×1 mL N132036-B 10×Dilution Buffer 1 mL 1 mL Storage Store at -25 to -15℃ in the dark. Valid for 1 year. Notes 1. For your safety and health, please wear a lab coat and disposable gloves when operating. 2. After thawing, the Master Mix may appear with flocculent or white precipitates. Hold it in hand and gently dissolve by slowly inverting it up and down until the solution is clear. This does not affect the performance of the reagent. 3. Before use, invert the Master Mix gently to mix. Do not vortex to avoid generating bubbles, which may affect the quantification results. The Master Mix can be used after mixing and brief centrifugation. During pipetting, gently aspirate and dispense. If bubbles form in the Master Mix due to improper operation, centrifuge it again before use. 4. Due to the extremely high sensitivity of this product, it is susceptible to aerosol contamination in the air. Therefore, when preparing the reaction mixture, please do it in a laminar flow cabinet. Use sterile pipette tips and reaction tubes during preparation. Laboratories with the conditions are recommended to use dedicated pipettes and filter-tipped pipette tips. 5. It is recommended to use our company's cDNA synthesis kit (Cat#N132062) to effectively remove residual genomic material from RNA samples. 6. For Research Use Only. Instructions 1. Template Processing The 2×Color qPCR Fluore Green Master Mix (No Rox) contains a blue dye, and the 10×Dilution Buffer is a dedicated yellow concentrated template dilution solution. During use, if template tracing is required, dilute it to 1X as the template dilution solution and then proceed with qPCR detection. If template tracing is not required, simply do not use the Dilution Buffer. Template Types Usage of 10× Dilution Buffer Final Concentration cDNA solution, dissolved plasmid, genome If you need to dilute the template, first dilute the template to the desired concentration with sterile ultrapure water. Then, add 1 μL of 10×Dilution Buffer to every 9 μL of the diluted template. 1× DNA Powder Dilute the 10×Dilution Buffer to 1× using sterile ultrapure water. Dissolve the corresponding DNA powder using an appropriate volume of 1× Dilution Buffer as the solvent. 1× 【Note】In actual use, other methods can also be employed as needed, as long as the final concentration of Dilution Buffer in the template is 1×. 2. Recommended qPCR Reaction System Component Volume（μL）*** Volume（μL）*** Final concentration 2×miRNA qPCR Master Mix 25 10 1× Forward Primer（self-provided）* 1 0.4 0.2 μM Reverse Primer（10 μM）* 1 0.4 0.2 μM cDNA ** x x - RNase-free H2O Up to 50 Up to 20 - 【Note】*The typical final concentration of primers is 0.2 μM, but it can also be adjusted between 0.1-1.0 μM depending on the situation.  **If the template is undiluted cDNA stock solution, the volume used should not exceed 1/10 of the total qPCR reaction volume. The optimal amount of template added should result in Ct values between 20-30 cycles for amplification. ***A reaction volume of 20 μL or 50 μL is recommended to ensure the effectiveness and reproducibility of target gene amplification. Mix thoroughly before running the reaction to avoid excessive bubbles from vigorous shaking. 3.   Reaction Program Recycling procedure Temperature Time Cycle Number Recycling procedure 95℃ 2 min 1 Pre-denaturation 95℃ 10 sec 40 Denaturation 60℃ 30 sec** Melting Curve Default Settings 1 【Note】*Annealing Temperature and Time: Please adjust according to the primer Tm value and the length of the target gene. **Fluorescence Signal Acquisition: Do not forget to enable fluorescence signal acquisition. Set up the experimental procedure according to the user manual of the instrument. ***Melting Curve: The default program of the instrument can usually be used. 4. Primer Design Guidelines 1) The recommended primer length is around 25 bp. The optimal amplicon size is 150 bp, but it can be chosen within the range of 100 bp to 300 bp. 2) The Tm values of the forward and reverse primers should not differ by more than 2℃. A Tm value between 60℃ and 65℃ is preferred. 3) The base distribution in the primer should be uniform, avoiding four consecutive identical bases. The GC content should be controlled around 50%. The last base at the 3’ end should preferably be G or C. 4) Avoid complementary sequences of more than three bases within a primer or between the forward and reverse primers. 5) The specificity of the primers should be verified using the NCBI BLAST program. Avoid having more than two non-specific complementary bases at the 3’ end of the primers. 6) The designed primers need to be tested for amplification efficiency. Only primers with the same amplification efficiency should be used for quantitative comparative analysis 5. Compatible Instruments This product contains a pre-mixed ROX Reference Dye 1 (high concentration) for correcting well-to-well fluorescence signal errors, and is suitable for the following real-time fluorescent quantitative PCR instruments: Applied Biosystems 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOnePlus; And other real-time fluorescent quantitative PCR instruments that require the addition of high-concentration ROX Reference Dye.

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Product description The 2×Color qPCR Fluore Green Master Mix (Low Rox) is a 2× real-time quantitative PCR amplification premix, characterized by high fluorescence intensity, high sensitivity, strong specificity, and high amplification yield. The core component, Taq DNA polymerase, utilizes an antibody-mediated hot-start mechanism, which effectively inhibits non-specific amplification caused by primer annealing during sample preparation. Additionally, the mix contains factors that enhance PCR amplification efficiency and promote balanced amplification across a wide range of GC content (30-70%), ensuring good linearity in quantitative PCR over a broad quantification range. This product employs a color-changing reaction based on the mixture of different dyes to monitor pipetting operations, effectively reducing the occurrence of pipetting errors. Specifications Catalog Number N132035E N132035S Specifications 50 T（20 μL/rxn） 250 T（20 μL/rxn） Components Component Identification Component Name N132034E N132034S N132035-A 2×Color qPCR Master Mix (Low Rox) 1 mL 5×1 mL N132035-B 10×Dilution Buffer 1 mL 1 mL Storage Store at -25 to -15℃ in the dark. Valid for 1 year. Notes 1. For your safety and health, please wear a lab coat and disposable gloves when operating. 2. After thawing, the Master Mix may appear with flocculent or white precipitates. Hold it in hand and gently dissolve by slowly inverting it up and down until the solution is clear. This does not affect the performance of the reagent. 3. Before use, invert the Master Mix gently to mix. Do not vortex to avoid generating bubbles, which may affect the quantification results. The Master Mix can be used after mixing and brief centrifugation. During pipetting, gently aspirate and dispense. If bubbles form in the Master Mix due to improper operation, centrifuge it again before use. 4. Due to the extremely high sensitivity of this product, it is susceptible to aerosol contamination in the air. Therefore, when preparing the reaction mixture, please do it in a laminar flow cabinet. Use sterile pipette tips and reaction tubes during preparation. Laboratories with the conditions are recommended to use dedicated pipettes and filter-tipped pipette tips. 5. It is recommended to use our company's cDNA synthesis kit (Cat#N132062) to effectively remove residual genomic material from RNA samples. 6. For Research Use Only. Instructions 1. Template Processing The 2×Color qPCR Fluore Green Master Mix (No Rox) contains a blue dye, and the 10×Dilution Buffer is a dedicated yellow concentrated template dilution solution. During use, if template tracing is required, dilute it to 1X as the template dilution solution and then proceed with qPCR detection. If template tracing is not required, simply do not use the Dilution Buffer. Template Types Usage of 10× Dilution Buffer Final Concentration cDNA solution, dissolved plasmid, genome If you need to dilute the template, first dilute the template to the desired concentration with sterile ultrapure water. Then, add 1 μL of 10×Dilution Buffer to every 9 μL of the diluted template. 1× DNA Powder Dilute the 10×Dilution Buffer to 1× using sterile ultrapure water. Dissolve the corresponding DNA powder using an appropriate volume of 1× Dilution Buffer as the solvent. 1× 【Note】In actual use, other methods can also be employed as needed, as long as the final concentration of Dilution Buffer in the template is 1×. 2. Recommended qPCR Reaction System Component Volume（μL）*** Volume（μL）*** Final concentration 2×miRNA qPCR Master Mix 25 10 1× Forward Primer（self-provided）* 1 0.4 0.2 μM Reverse Primer（10 μM）* 1 0.4 0.2 μM cDNA ** x x - RNase-free H2O Up to 50 Up to 20 - 【Note】*The typical final concentration of primers is 0.2 μM, but it can also be adjusted between 0.1-1.0 μM depending on the situation.  **If the template is undiluted cDNA stock solution, the volume used should not exceed 1/10 of the total qPCR reaction volume. The optimal amount of template added should result in Ct values between 20-30 cycles for amplification. ***A reaction volume of 20 μL or 50 μL is recommended to ensure the effectiveness and reproducibility of target gene amplification. Mix thoroughly before running the reaction to avoid excessive bubbles from vigorous shaking. 3.   Reaction Program Recycling procedure Temperature Time Cycle Number Recycling procedure 95℃ 2 min 1 Pre-denaturation 95℃ 10 sec 40 Denaturation 60℃ 30 sec** Melting Curve Default Settings 1 【Note】*Annealing Temperature and Time: Please adjust according to the primer Tm value and the length of the target gene. **Fluorescence Signal Acquisition: Do not forget to enable fluorescence signal acquisition. Set up the experimental procedure according to the user manual of the instrument. ***Melting Curve: The default program of the instrument can usually be used. 4. Primer Design Guidelines 1) The recommended primer length is around 25 bp. The optimal amplicon size is 150 bp, but it can be chosen within the range of 100 bp to 300 bp. 2) The Tm values of the forward and reverse primers should not differ by more than 2℃. A Tm value between 60℃ and 65℃ is preferred. 3) The base distribution in the primer should be uniform, avoiding four consecutive identical bases. The GC content should be controlled around 50%. The last base at the 3’ end should preferably be G or C. 4) Avoid complementary sequences of more than three bases within a primer or between the forward and reverse primers. 5) The specificity of the primers should be verified using the NCBI BLAST program. Avoid having more than two non-specific complementary bases at the 3’ end of the primers. 6) The designed primers need to be tested for amplification efficiency. Only primers with the same amplification efficiency should be used for quantitative comparative analysis 5. Compatible Instruments This product contains a pre-mixed ROX Reference Dye 2 (low concentration) for correcting well-to-well fluorescence signal errors, and is suitable for the following real-time fluorescent quantitative PCR instruments: Applied Biosystems 7500, 7500 Fast, ViiA7; Stratagene MX4000, MX3005P, MX3000P; And other real-time fluorescent quantitative PCR instruments that require the addition of low-concentration ROX Reference Dye.

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Product description The 2×Color qPCR Fluore Green Master Mix (No Rox) is a 2× real-time quantitative PCR amplification premix, characterized by high amplification yield, high fluorescence intensity, high sensitivity, and strong specificity. The core component, Taq DNA polymerase, utilizes an antibody-mediated hot-start mechanism, which effectively inhibits non-specific amplification caused by primer annealing during sample preparation. Additionally, the mix contains factors that enhance PCR amplification efficiency and promote balanced amplification across a wide range of GC content (30-70%), ensuring good linearity in quantitative PCR over a broad quantification range. This product employs a color-changing reaction based on the mixture of different dyes to monitor pipetting operations, effectively reducing the occurrence of pipetting errors. Specifications Catalog Number N132034E N132034S Specifications 50 T（20 μL/rxn） 250 T（20 μL/rxn） Components Component Identification Component Name N132034E N132034S N132034-A 2×Color qPCR Master Mix (No Rox) 1 mL 5×1 mL N132034-B 10×Dilution Buffer 1 mL 1 mL Storage Store at -25 to -15℃ in the dark. Valid for 1 year. Notes 1. For your safety and health, please wear a lab coat and disposable gloves when operating. 2. After thawing, the Master Mix may appear with flocculent or white precipitates. Hold it in hand and gently dissolve by slowly inverting it up and down until the solution is clear. This does not affect the performance of the reagent. 3. Before use, invert the Master Mix gently to mix. Do not vortex to avoid generating bubbles, which may affect the quantification results. The Master Mix can be used after mixing and brief centrifugation. During pipetting, gently aspirate and dispense. If bubbles form in the Master Mix due to improper operation, centrifuge it again before use. 4. Due to the extremely high sensitivity of this product, it is susceptible to aerosol contamination in the air. Therefore, when preparing the reaction mixture, please do it in a laminar flow cabinet. Use sterile pipette tips and reaction tubes during preparation. Laboratories with the conditions are recommended to use dedicated pipettes and filter-tipped pipette tips. 5. It is recommended to use our company's cDNA synthesis kit (Cat#N132062) to effectively remove residual genomic material from RNA samples. 6. For Research Use Only. Instructions 1. Template Processing The 2×Color qPCR Fluore Green Master Mix (No Rox) contains a blue dye, and the 10×Dilution Buffer is a dedicated yellow concentrated template dilution solution. During use, if template tracing is required, dilute it to 1X as the template dilution solution and then proceed with qPCR detection. If template tracing is not required, simply do not use the Dilution Buffer. Template Types Usage of 10× Dilution Buffer Final Concentration cDNA solution, dissolved plasmid, genome If you need to dilute the template, first dilute the template to the desired concentration with sterile ultrapure water. Then, add 1 μL of 10×Dilution Buffer to every 9 μL of the diluted template. 1× DNA Powder Dilute the 10×Dilution Buffer to 1× using sterile ultrapure water. Dissolve the corresponding DNA powder using an appropriate volume of 1× Dilution Buffer as the solvent. 1× 【Note】In actual use, other methods can also be employed as needed, as long as the final concentration of Dilution Buffer in the template is 1×. 2. Recommended qPCR Reaction System Component Volume（μL）*** Volume（μL）*** Final concentration 2×miRNA qPCR Master Mix 25 10 1× Forward Primer（self-provided）* 1 0.4 0.2 μM Reverse Primer（10 μM）* 1 0.4 0.2 μM cDNA ** x x - RNase-free H2O Up to 50 Up to 20 - 【Note】*The typical final concentration of primers is 0.2 μM, but it can also be adjusted between 0.1-1.0 μM depending on the situation.  **If the template is undiluted cDNA stock solution, the volume used should not exceed 1/10 of the total qPCR reaction volume. The optimal amount of template added should result in Ct values between 20-30 cycles for amplification. ***A reaction volume of 20 μL or 50 μL is recommended to ensure the effectiveness and reproducibility of target gene amplification. Mix thoroughly before running the reaction to avoid excessive bubbles from vigorous shaking. 3.   Reaction Program Recycling procedure Temperature Time Cycle Number Recycling procedure 95℃ 2 min 1 Pre-denaturation 95℃ 10 sec 40 Denaturation 60℃ 30 sec** Melting Curve Default Settings 1 【Note】*Annealing Temperature and Time: Please adjust according to the primer Tm value and the length of the target gene. **Fluorescence Signal Acquisition: Do not forget to enable fluorescence signal acquisition. Set up the experimental procedure according to the user manual of the instrument. ***Melting Curve: The default program of the instrument can usually be used. 4. Primer Design Guidelines 1) The recommended primer length is around 25 bp. The optimal amplicon size is 150 bp, but it can be chosen within the range of 100 bp to 300 bp. 2) The Tm values of the forward and reverse primers should not differ by more than 2℃. A Tm value between 60℃ and 65℃ is preferred. 3) The base distribution in the primer should be uniform, avoiding four consecutive identical bases. The GC content should be controlled around 50%. The last base at the 3’ end should preferably be G or C. 4) Avoid complementary sequences of more than three bases within a primer or between the forward and reverse primers. 5) The specificity of the primers should be verified using the NCBI BLAST program. Avoid having more than two non-specific complementary bases at the 3’ end of the primers. 6) The designed primers need to be tested for amplification efficiency. Only primers with the same amplification efficiency should be used for quantitative comparative analysis 5. Compatible Instruments This product does not include ROX Reference Dye for correcting well-to-well fluorescence signal errors. It is compatible with the following real-time fluorescent quantitative PCR instruments: Bio-Rad CFX96, CFX384, iCycler iQ, iQ5, MyiQ; MiniOpticon, Opticon, Opticon 2, Chromo4; Cepheid SmartCycler; Eppendorf Mastercycler ep realplex, realplex 2 s; Illumina Eco qPCR; Qiagen/Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000; Roche Applied Science LightCycler 480; Thermo Scientific PikoReal Cycler; and other real-time fluorescent quantitative PCR instruments that do not require the addition of ROX Reference Dye.Dye-based qPCRdye-based

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Product description 2×Universal qPCR Fluore Green Master Mix (No ROX adjustment) is a pre-mixed solution for 2× real-time quantitative PCR amplification, featuring high sensitivity and specificity. It is blue in color and serves as a loading tracer. The core component, Taq DNA polymerase, is thermally activated by an antibody method, effectively suppressing non-specific amplification caused by primer annealing during sample preparation. Additionally, it contains factors that enhance PCR amplification efficiency and promote the amplification of genes with different GC contents (30-70%), ensuring a good linear relationship within a broad quantitative range. This product contains a special ROX Passive Reference Dye and is suitable for all qPCR instruments without the need for ROX adjustment based on different instruments. Specifications Catalog Number N132031E N132031S N132031M N132031L Specifications 50 T（20 μL/rxn） 250 T（20 μL/rxn） 2500 T（20 μL/rxn） 5000 T（20 μL/rxn） Storage Ice bag transportation. Store at -20℃ in the dark. Valid for 18 months. This product should avoid repeated freezing and thawing. The product contains the fluorescent dye SYBR Green I. When storing or preparing the reaction system, strong light exposure should be avoided. Notes 1. It is recommended to use Arcegen One-Step cDNA Synthesis RT Premix (Cat#N132061) to effectively remove residual genomic DNA from RNA samples. 2. After thawing, Master Mix may present flocculent or white precipitates. Hold it in your hand to slowly dissolve it and gently invert it up and down to mix until the solution becomes clear. This does not affect the performance of the reagent. 3. For your safety and health, please wear a lab coat and disposable gloves when operating. 4. This product is for research use only. Instructions 1. Reaction System Component volume（μL） volume（μL） Final concentration 2×qPCR Master Mix 25 10 1× Forward Primer (10 μM) 1 0.4 0.2 μM Reverse Primer (10 μM) 1 0.4 0.2 μM Template DNA X X - Sterile ultrapure water to 50 to 20 - 【Note】Before use, make sure to mix thoroughly to avoid vigorous shaking that may produce excessive bubbles. 1) Primer concentration: The typical final concentration of primers is 0.2 μM, but it can also be adjusted between 0.1-1.0 μM depending on the situation. 2) Template concentration: If the template is undiluted cDNA stock solution, the volume used should not exceed 1/10 of the total qPCR reaction volume. 3) Template dilution: It is recommended to dilute the cDNA stock solution 5-10 times. The optimal amount of template added should result in Ct values between 20-30 cycles for amplification. 4) Reaction volume: A reaction volume of 20 μL or 50 μL is recommended to ensure the effectiveness and reproducibility of target gene amplification. 5) Reaction set up: Please prepare the reaction mixture inside a laminar flow cabinet and use nuclease-free pipette tips and reaction tubes. Filter-tipped pipette tips are recommended. Avoid cross-contamination and aerosol contamination. Standard Procedure Recycling Procedure  Temperature Time Cycle Number Pre-denaturation 95℃ 2 min 1 Denaturation 95℃ 10 sec 40 Annealing/extension 60℃ 30 sec★ Melting Curve Analysis Default Settings 1 Fast Program Recycling Procedure  Temperature Time Cycle Number Pre-denaturation 95℃ 30 sec 1 Denaturation 95℃ 3 sec 40 Annealing/extension 60℃ 20 sec★ Melting Curve Analysis Default Settings 1 【Note】The fast program is applicable to the vast majority of genes. For genes with complex secondary structures, the standard program can be attempted. 1) Annealing temperature and time: Please adjust according to the length of the primers and the target gene. 2) Fluorescence signal acquisition (★): Do not forget to enable fluorescence signal acquisition. Set up the experimental procedure according to the user manual of the instrument. The time settings for several common instruments are as follows: 20 sec: Applied Biosystems 7700, 7900HT, 7500 Fast 31 sec: Applied Biosystems 7300 32 sec: Applied Biosystems 7500 3) Melting curve: The default program of the instrument can usually be used. 2. Data Analysis Quantitative experiments should include at least three biological replicates. After the reaction, it is necessary to confirm the amplification curves and melting curves. 1) Amplification Curves: The standard amplification curve should be S-shaped. The most accurate quantitative analysis is achieved when the Ct value falls between 20 and 30. If the Ct value is less than 10, the template should be diluted and the experiment should be repeated. If the Ct value is between 30 and 35, the template concentration should be increased, or the reaction volume should be enlarged to improve amplification efficiency and ensure the accuracy of the results. If the Ct value is greater than 35, the results cannot be used for quantitative analysis of gene expression but can be used for qualitative analysis. 2) Melting Curves: A single peak in the melting curve indicates good specificity of the reaction and allows for quantitative analysis. If the melting curve shows a double peak or multiple peaks, quantitative analysis cannot be performed. If a double peak appears in the melting curve, DNA agarose gel electrophoresis should be used to determine whether the non-target peak is due to primer dimers or non-specific amplification. If it is a primer dimer, it is recommended to reduce the primer concentration or redesign primers with higher amplification efficiency. If it is non-specific amplification, the annealing temperature should be increased, or primers with higher specificity should be redesigned. 3. Primer Design Guidelines 1) The recommended primer length is around 25 bp. The optimal length of the amplicon is 150 bp, but it can be chosen within the range of 100 bp to 300 bp. 2) The Tm values of the forward and reverse primers should not differ by more than 2℃. A Tm value between 60℃ and 65℃ is preferred for primers. 3) The base distribution in the primer should be uniform, avoiding four consecutive identical bases. The GC content should be controlled around 50%. The last base at the 3’ end should preferably be G or C. 4) It is best to avoid complementary sequences of more than three bases within a primer or between the forward and reverse primers. 5) The specificity of the primers should be verified using the NCBI BLAST program. Avoid having more than two non-specific complementary bases at the 3’ end of the primers. 6) The designed primers need to be tested for amplification efficiency. Only primers with the same amplification efficiency should be used for quantitative comparative analysis. 4. Compatible Instruments ABI: 5700, 7000, 7300, 7700, 7900HT Fast, StepOne, StepOne Plus; 7500, 7500 Fast, ViiA7, QuantStudio 3 and 5, QuantStudio 6,7,12k Flex; Stratagene: MX3000P, MX3005P, MX4000P;   Bio-Rad: CFX96, CFX384, iCycler iQ, iQ5, MyiQ, MiniOpticon, Opticon, Opticon 2, Chromo4; Eppendorf: Mastercycler ep realplex, realplex 2 s; Qiagen: Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000; Roche Applied Science: LightCycler 480, LightCycler 2.0; Lightcycler 96; Thermo Scientific: PikoReal Cycler; Cepheid: SmartCycler; Illumina: Eco qPCR.

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