Dye-based RT-qPCR

Product description The Universal One Step RT-qPCR Fluore Green Kit is a reagent kit for fluorescent quantitative detection based on SYBR Green I dye. Using gene-specific primers, reverse transcription and qPCR reactions are completed in a single tube, eliminating the need for repeated opening and pipetting, which greatly improves detection efficiency and reduces the risk of contamination. The buffer system has been optimized. For highly expressed targets, the kit's sensitivity can reach 0.1 pg, and for moderately expressed targets, it can reach 1 pg. This kit is also suitable for amplification and quantification of DNA samples. The kit can achieve highly sensitive detection and quantification of nucleic acids from various animal and plant samples, cells, and microorganisms. Specifications Catalog Number N132051E N132051S Specifications 20 T（20 μL/rxn） 200T（20 μL/rxn） Components Component Identification Component Name N132051E N132051S N132051-A 2× Universal SG Buffer 250 μL 2×1.25 mL N132051-B Universal UH Enzyme Mix 20 μL 200 μL N132051-C RNase free H2O 250 μL 2×1.25 mL Storage Store at -25 to -15℃ in the dark. Valid for 1 year. Notes 1. All steps for sample addition and solution preparation should be performed on ice whenever possible. 2. Each component should be vortexed to mix thoroughly and then briefly centrifuged at low speed before use. 3. For your safety and health, please wear a lab coat and disposable gloves when operating. 4. For Research Use Only. Instructions 1. Recommended Reaction System Component Volume（μL）**** Volume（μL） Final concentration 2× Universal SG Buffer 12.5 25 1× Universal UH Enzyme Mix 1 2 - Forward Primer (10 μM)** 0.5 1 0.2 μM Reverse Primer (10 μM)** 0.5 1 0.2 μM Template RNA*** X X   RNase-free ddH2O to 25 to 50   **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. ***This reagent is highly sensitive. For Total RNA in the range of 1 pg – 1 μg, tests with human samples show that the optimal input amount is 1 pg – 100 ng, with Ct values ideally falling between 15 and 30. ****A reaction volume of 20 μL or 50 μL is recommended to ensure the effectiveness and reproducibility of target gene amplification. *****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. 2. Reaction Program Recycling procedure Temperature Time Cycle Number Recycling procedure 50℃* 6 min 1 Pre-denaturation 95℃ 5 min 1 Denaturation 95℃ 15 sec 40 60℃** 30 sec Melting Curve Default Settings 1 The reverse transcription temperature can be selected between 50-55℃ according to experimental requirements. For DNA samples, the reverse transcription step can be omitted. ** In special cases, the annealing/extension temperature can be adjusted according to the primer Tm value, with 60℃ being recommended. 3. Compatible Instruments Instrument models that do not require ROX calibration: 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, LightCycler2.0, Lightcycler 96; Thermo Scientific: PikoReal Cycler; Cepheid: SmartCycler; Illumina: Eco qPCR; Low Rox： ABI 7500, 7500 Fast, ViiA7, QuantStudio 3 and 5, QuantStudio 6, 7, 12k Flex; Stratagene MX3000P, MX3005P, MX4000P; High Rox： ABI 5700, 7000, 7300, 7700, 7900HT Fast, StepOne, StepOne Plus. 4. Primer Design Tips 1) Primers should ideally be designed to span an exon-exon junction, with one of the amplification primers potentially crossing the actual exon-intron boundary. This design can reduce the risk of amplifying false positives from contaminated genomic DNA. 2) Primers should be specific. After primer design is completed, a BLAST search should be performed to check for specificity. 3) Primer length is generally between 18 and 27 bp. Primers should not be too long, as this can lead to high extension temperatures that are not suitable for Taq DNA polymerase reactions. 4) The GC content of primers should be between 40% and 60%. Both too high and too low GC content are not conducive to the reaction. The GC content of the forward and reverse primers should not differ significantly. Additionally, the Tm value of the primers, which is the melting temperature of the oligonucleotide, is the temperature at which 50% of the oligonucleotide duplexes are dissociated under a given salt concentration. The effective annealing temperature is generally 5-10℃ higher than the Tm value. The Tm value of the primer can be estimated using the formula Tm = 4(G+C) + 2(A+T), or it can be calculated using software. 5) The length of the PCR product is usually controlled between 80 and 300 bp. To ensure amplification efficiency, the length of the PCR product should be considered when designing primers. 6) The 3′ end of the primer should avoid having an A. When there is a mismatch at the 3′ end of the primer, there is a significant difference in the initiation efficiency of different bases. When the last base is A, chain synthesis can occur even in the case of a mismatch. However, when the last base is T, the mismatch initiation efficiency is greatly reduced. The initiation efficiency of G and C mismatches is between that of A and T. Therefore, it is best to choose T at the 3′ end. 7) Bases should be randomly distributed, and there should be no runs of purines or pyrimidines. In particular, there should not be more than three consecutive Gs or Cs at the 3′ end, as this can cause the primer to initiate incorrectly in GC-rich sequence regions. 8) Primers should avoid complementary sequences within themselves and between each other. Otherwise, the primer may fold into a hairpin structure, causing the primer to anneal to itself. Such secondary structures can hinder the annealing of the primer to the template due to steric hindrance. Primers should not have more than four consecutive complementary bases within themselves. There should also be no complementarity between the two primers, especially at the 3′ ends, to prevent the formation of primer dimers. There should not be more than four consecutive complementary bases between primers. If primer dimers and hairpin structures are unavoidable, their DG values should be kept low (less than 4.5 kcal/mol). Otherwise, they can easily lead to the formation of primer dimer bands and reduce the effective concentration of primers, preventing the PCR reaction from proceeding normally. 5. Analysis of Abnormal Results 1) No Ct Value Detected Incorrect Fluorescence Signal Collection: Ensure that the fluorescence signal is collected at the end of the annealing/extension step. Primer Degradation: Check the integrity of the primers using PAGE electrophoresis. Insufficient Template Amount: For samples of unknown concentration, start testing from the original solution. Template Degradation: Avoid introducing impurities during sample preparation and prevent repeated freeze-thaw cycles. 2) High Ct Value (Ct > 35) Low Amplification Efficiency: Optimize reaction conditions and check primer design. Consider using a three-step reaction method or slightly lowering the annealing temperature. Too Long PCR Product: Generally, use a product length of 80-150 bp. 3) Poor Linearity of Standard Curve . Pipetting Errors: This can cause the standards not to form a gradient. For RNA templates, it is recommended to use 1× TE buffer for gradient dilution, while for DNA templates, ddH2O is recommended for dilution. Standard Degradation: Avoid repeated freeze-thaw cycles of the standards, or re-prepare and dilute the standards. Poor Primer Design: Redesign the primers. Presence of Inhibitors in the Template or Excessive Template Concentration. 4) NTC (No Template Control) Amplification (Ct < 32) Suboptimal Primer Design: Avoid primer dimers and hairpin structures. Primer Concentration Issues: Adjust the primer concentration and ensure the proper ratio between the forward and reverse primers. Aerosol Contamination: Leakage of amplified products can easily cause aerosol contamination in the laboratory, which can lead to inaccurate quantitative detection results once contamination occurs.

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