FastSYBR Mixture (C09-1539-644)

Product content:Component:F665743-5ml:2×FastSYBR Mixture:5×1 ml:50×Low ROX:200 μl:ddH2O:5×1 ml:Product Introduction:FastSYBR Mixture is a premixed system for dye-based (SYBR Green I) real-time fluorescent quantitative PCR, with a concentration of 2×, containing Fast Taq DNA Polymerase, PCR Buffer, dNTPs, SYBR Green I fluorescent dye and Mg2+, which is easy to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The fluorescent dye SYBR Green I contained in this product can bind to all double-stranded DNAs, allowing the product to be used for the detection of different target sequences without the need to synthesize specific labeling probes. The Fast Taq DNA Polymerase can effectively reduce the non-specific amplification caused by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only requires incubation at 95℃ for 20 s. The whole PCR reaction can save about 40 minutes compared with the normal reaction, which greatly shortens the reaction time of PCR. The unique combination of PCR buffer system and hot-start enzyme effectively inhibits the generation of non-specific products and significantly improves the amplification efficiency of PCR. The product has a wide range of applications and is suitable for both normal and rapid quantitative PCR programs.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration:Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments requiring Low ROX calibration:ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments requiring High ROX calibration:ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention:1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. This product contains the fluorescent dye SYBR Green I. Avoid strong light when storing this product or preparing PCR reaction solution.3. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.4. This product cannot be used for fluorescent quantitative PCR by the probe method.Usage:The following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction syste:Reagents:50 μlReaction system:final concentration:2×FastSYBR Mixture:25 μl:1×:Forward Primer，10 µM:1 μl:0.2 μM ¹⁾:Reverse Primer，10 µM:1 μl:0.2 μM ¹⁾:Template DNA:2 μl ²⁾: :50×Low ROX or High ROX（optional）³⁾:1 μl:1 ×:ddH2O:up to 50 μl:Note: 1) Typically, the primer concentration is 0.2 μ M can achieve good results, ranging from 0.1 to 1.0 μ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased: When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.2) The amount of DNA template is usually based on 10-100 ng genomic DNA or 1-10 ng cDNA as a reference. Due to the different copy numbers of target genes contained in templates of different species, gradient dilution can be applied to the template to determine the optimal template usage.3) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added:2. PCR reaction conditions:Note: 1) The enzyme used in this product must be pre-denatured at 95°C and 20s to achieve enzyme activation. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1 minute to allow the starting template to fully unchain. If the high temperature treatment time is too long, it will affect the enzyme activity. The optimal pre-denaturation time can be determined according to the template.(2) It is recommended to use two-step PCR reaction program, the annealing temperature should be 60-64 ℃ as the reference of the setting range, and the annealing temperature can be increased when a non-specific reaction occurs. If you can't get good results due to the use of primers with low Tm value, you can try three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference.(3) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument used, and this program is set up with the ABI 7500 fluorescence quantitative PCR instrument as a reference. Specifications and Purity: Low ROX