Probe-based qPCR

Product description This reagent is a 2× qPCR premix system that supports the completion of up to four-channel fluorescence quantitative PCR detection in a single reaction well. The core of the product features a high-performance antibody-mediated hot-start Taq DNA polymerase, which has been genetically engineered to significantly enhance the detection sensitivity and target specificity of the amplification reaction. Specifically optimized for multiplex PCR reactions, the buffer system formulation effectively enhances nucleic acid amplification efficiency, particularly improving the amplification success rate of low-concentration samples. This kit is suitable for molecular detection fields such as gene polymorphism analysis and multiplex target quantitative detection. Specifications Catalog Number N132041E N132041S N132041M Specifications 80 T（12.5 μL/rxn） 400 T（12.5 μL/rxn） 1600 T（12.5 μL/rxn） Components Component Identification Component Name N132041E N132041S N132041M N132041 2×Multiplex qPCR Probe Master Mix 1 mL 5×1 mL 20 mL Storage Store at -25 to -15℃. Valid for 2 years. Notes 1. Please prepare the reaction system inside a laminar flow hood and use pipette tips and reaction tubes that are free of nucleic acid residues. It is recommended to use pipette tips with filters to avoid cross-contamination and aerosol contamination. 2. For your safety and health, please wear a lab coat and disposable gloves when operating. 3. This product is for research use only. Instructions 1. Reaction System   Component Volume（μL） Final concentration 2×Multiplex qPCR Probe Master Mix 12.5 1× Primer mix (10 μM) X 0.1 μM-0.5 μM Probe mix (10 μM) X 50 nM-250 nM Rox reference dye 0.5 1× Template DNA/cDNA 1-10 - ddH2O Up to 25 - 【Notes】 1) Mix thoroughly before use to avoid generating excessive bubbles from vigorous shaking. 2) The Primer Mix contains multiple pairs of primers, with each primer typically at a final concentration of 0.2 µM, which can also be adjusted between 0.1-0.5 µM as needed. 3) The Probe Mix contains multiple probes with different fluorescent signals, and the concentration of each probe can be adjusted between 50-250 nM as required. 4) This product does not contain Rox reference dye. If needed, it is recommended to use the product with catalog number N132049. 5) qPCR is highly sensitive. It is recommended to dilute the template before use. If the template is undiluted cDNA, the template volume should not exceed 1/10 of the total reaction volume. 6) Reaction System: It is recommended to use a reaction volume of 25 µL, 30 µL, or 50 µL to ensure the effectiveness and reproducibility of target gene amplification. 2. Recommended Amplification Protocol Recycling procedure Temperature Time Cycle Number Pre-denaturation 95℃ 5 min 1 Denaturation 95℃ 15 sec 45 Annealing/Extension 60℃ 30 sec 【Note】 1) Amplification Reaction: The amplification reaction temperature should be adjusted according to the Tm value of the designed primers. 2) Fluorescence Signal Acquisition: The required fluorescence signal acquisition time varies among different qPCR instruments. Please set according to the shortest time limit.   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. 3.  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. 4. Compatible Instruments Applied Biosystems: 5700, 7000, 7300, 7700, 7900HT Fast, StepOne™ , StepOne Plus™ , 7500, 7500 Fast, ViiA™ 7, QuantStudio™ 3 and 5, QuantStudio™ 6,7,12k Flex; 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; Stratagene: MX3000P™, MX3005P™, MX4000P™; Thermo Scientific: PikoReal Cycler; Cepheid: SmartCycler; Illumina: Eco qPCR; SLAN: SLAN-96S,SLAN-96P.

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