| Product Description | Aladdin's UltraBio™ Probe qPCR Mix (2X, Low ROX) is a high-quality supermix for probe-based real-time PCR which is also called TaqMan probe-based qPCR when TaqMan probe is used. It is mainly used in gene expression studies and genomics applications, with high specificity and sensitivity.This product is particularly suitable for quantitative or qualitative analysis of target RNAs with low abundance or high specificity, such as lncRNA, sRNA, low abundance mRNA and microbial RNA. It can also be used to differentiate the expression level of SNPs or some homologous genes with high similarity, which usually could not be achieved using the traditional dye method.The specificity and sensitivity of this kit are superior to that of SYBR Green qPCR Mix. A fluorescent signal is generated by the specific binding with target DNA and the following degradation of the probe. Therefore, the specificity of PCR amplification is ensured by both PCR primers and the probe.This product can be used for multiplex PCR to detect the expression level of multiple genes in a single reaction tube. Different probes with different fluorescence representing different genes are added in a single reaction mixture. Using this kit, two to three genes can be quantified simultaneously in a single reaction mixture with optimized primers and probes.The probe-based real-time PCR uses an oligonucleotide probe complementary to an internal segment of the amplified target DNA. The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps the excitation spectrum of the other, resulting in the “quenching” of the first fluorophore by the second. During the PCR amplification, both the primers and probes are annealed to the target DNA. With the extension of the primers, the Taq DNA polymerase with 5'→3' exonuclease activity degrades the oligonucleotide probe gradually from the 5' end, releasing the fluorophore group that can be excited by excitation light to produce fluorescence. During PCR, as the target sequence is amplified, more fluorescent groups will be released. The fluorescence intensity is proportional to the number of PCR amplicons, by which the amount of dsDNA generated by PCR amplification can be quantified by measuring the fluorescence intensity. Generally, the 5' end of an oligonucleotide probe is labeled with fluorophore groups such as FAM or HEX and the 3' end is labeled with fluorescence quenchers such as BHQ1, TAMRA or MGB.This product 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™ Probe qPCR Mix (2X, Low ROX) is a 2X concentrated solution of UltraBio™ Taq DNA Polymerase, dNTPs, and all of the components required for PCR, except probes, DNA template and primers.This product contains a low concentration of ROX and can be used on qPCR instruments requiring Low 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™ Probe 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-Rad
Precautions： 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 Probe qPCR Mix (2X, Low ROX). Once melted, keep it on ice.b. Assemble the qPCR reaction on ice or at room temperature as follows (e.g., in a 96-well plate):ComponentVolume (µl) Probe qPCR Mix (2X, Low ROX)10 µlForward and Reverse Primer Mix (3μM each)2 µlProbe0.5 µlTemplate DNA2 µlRNase-free Waterto 50 µlNote 1: Each 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: The optimal concentration of probe is dependent on the type of qPCR instrument, probe, and the fluorophore group. In general, the concentration of probe should be lower than primer concentration. For example, if the primer concentration is 0.3 μM, the concentration of probe is recommended to be 0.2 μM, which can be adjusted appropriately in the range of 0.1-0.3 μM.Note 3: 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 4: A reaction volume of 20µl is recommended for each well of a 96-well plate, which can be adjusted based on your requirements.Note 5: 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 60ºC, 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.
|
|---|
