UltraBioâ„¢ SYBR Green qPCR Mix (C007B-566170)

Aladdin's UltraBio™ SYBR Green qPCR Mix (2X) is a high-quality supermix for real-time PCR. It is mainly used in gene expression studies and genomics applications, with high specificity and sensitivity.SYBR Green I contained in the UltraBio™ SYBR Green qPCR Mix (2X) is a dye with green fluorescence that binds into the minor groove of dsDNA. It has very weak fluorescence in the free state, but when binding to dsDNA, its fluorescence is increased significantly. During PCR, as the target sequence is amplified, SYBR Green I binds to each new copy of dsDNA during the extension step, by which the amount of dsDNA generated by PCR amplification can be quantified by measuring the fluorescence intensity.UltraBio™ SYBR Green qPCR Mix (2X) contains UltraBio™ Taq DNA Polymerase and its monoclonal antibody that binds with DNA polymerase and inhibits its activity at room temperature, allowing reactions to be set up at room temperature. During the initial DNA denaturation step (reaction temperature ~94°C), the antibody is denatured, releasing the polymerase and allowing DNA synthesis to proceed. The use of a hot-start DNA polymerase prevents nonspecific amplification due to mispriming and/or the formation of primer dimers during PCR assembly at lower temperatures, thus greatly improving the specificity, sensitivity and accuracy of qPCR reactions.UltraBio™ SYBR Green qPCR Mix (2X) is a 2X concentrated solution of Taq DNA Polymerase, dNTPs, SYBR Green I, and all of the components required for PCR, except DNA template and primers.This product does not contain ROX fluorescent dye and can be used on qPCR instrument that does not require ROX as a reference dye. ROX is usually used as a passive reference fluorescent dye in qPCR for signal normalization. ROX is not essential for qPCR, however, adding it can help reduce variations of fluorescent values in samples induced by pipetting errors, sample evaporation, or other technical problems. The optimal ROX concentration in qPCR reaction is dependent on the type of qPCR instrument. Based on the type of qPCR instrument available, choose High ROX, Low ROX or ROX-free qPCR mix. The UltraBio™ SYBR Green qPCR Mix (2X, High ROX) can also be used on qPCR instruments requiring no or low concentrations of ROX. Refer to the following table for qPCR instruments with different requirements of ROX.ROX RequirementReal-time PCR InstrumentNo RequirementBio-RadPrecautions：PrecautionsPrecautions should be taken to avoid cross-contamination. Discard PCR products after sealing to avoid their contamination of experimental surroundings.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. Prepare qPCR reactions:a. Thaw and mix well the SYBR Green qPCR Mix (2X). Once melted, keep it on ice.b. Assemble qPCR reactions on ice as follows (e.g., in a 96-well plate):ComponentVolume (µl) SYBR Green qPCR Mix (2X)10Forward and Reverse Primer Mix (3μM each)2Template DNA2RNase-free Water6Total Volume20Note 1: Primer at a final concentration of 0.2-0.5μM normally works well, but primer concentration can be optimized between 0.1-1.0 μM. Note 2: Add 1-10ng cDNA or 10-100ng genomic DNA in 20 µl of qPCR reaction as a starting point. When necessary, the DNA templates can be diluted in a gradient manner to determine the optimal amount of DNA template for qPCR reaction. When the cDNA obtained by RT-PCR reaction is directly used as the template, the addition amount should not exceed 10% of the total volume of PCR reaction. Note 3: A reaction volume of 20µl is recommended for each well of a 96-well plate, which can be adjusted as needed.Note 4: Negative control without DNA template is always recommended.c. Mix well reactions by gentle vortex or pipetting. Centrifuge briefly to collect the liquid at the bottom of the PCR tube.2. Transfer qPCR reactions to a qPCR instrument and run thermocycling conditions as follows:StepTemperatureDurationCyclesInitial Denaturation95℃2 min1Denaturation95℃15 sec40Annealing and Extension60℃15-30 secMelting Curve (optional)95℃15 sec160℃15 sec195℃15 sec1Note 1: The PCR running conditions listed above are for general use only. They can be adjusted based on the template, primer sequence, the length of PCR product or GC content, etc. Note 2: The duration for initial denaturation is usually 2 min, but can be adjusted to 5 min for the amplification of complex or GC-rich DNA fragments.Note 3: The Taq DNA Polymerase is capable of amplifying at least 300bp in 15 seconds. For amplicons longer than 350bp or with high GC content, it is recommended to increase the extension time to 60 s or to use a three-step method to improve amplification efficiency.Note 4: When the annealing temperature of primers is lower than 60℃, we recommend using the three-step method for PCR amplification. 3. qPCR data analysis using the software provided in the qPCR instrument.FAQ:1. Nonspecificity or low amplification efficiency.a. Primer sequence is not well designed. Use primer design tools for primer design to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. Use primers published in the literature or order the tested qPCR primers from .b. The targeted DNA fragments may have a high GC content. It is difficult to amplify DNA fragments with high GC content. Redesign primers at a different region of the DNA target.c. Preparation of PCR reaction mixture at room temperature results in nonspecific amplification easily, which can be prevented by using the hot-start DNA polymerase. To amplify DNA fragment which is hard to be amplified, assembling the PCR reaction mixture on ice can further reduce the nonspecific amplification.d. Annealing temperature needs to be optimized or redesign primers.e. The denaturation is insufficient for DNA amplicons with high GC content or longer length. In this case, redesign primers on regions easier to be amplified.f. The amount of DNA templates is too low. Add more DNA templates.g. DNA templates contain substances that inhibit PCR reactions and can be purified using appropriate DNA purification methods such as column purification.2. Optimization of PCR conditions.a. Primers at a final concentration of 0.2-0.5μM normally work well, but primer concentration can be adjusted between 0.1-1.0 μM. Reduce primer concentration to improve the specificity of PCR amplification and increase primer concentration to improve the amplification efficiency.b. Annealing temperature: Two-step PCR amplification is regularly used with annealing and extension temperature at 60ºC. The annealing/extension temperature can be adjusted in the range of 60-64ºC. The specificity of qPCR reactions can be improved by increasing the annealing temperature appropriately. When the Tm value of primers is low, three-step PCR amplification can be tried. The annealing temperature of the three-step method is recommended to be between 56-64ºC.c. Extension time: Two-step PCR amplification is recommended with an extension time between 15-30 seconds. For amplicons longer than 350bp or with high GC content, it is recommended to increase the extension time to 60 s or to use a three-step amplification method to improve amplification efficiency.

Specification: 2X