Quantitative PCR (qPCR, also known as Real-time PCR) is a very versatile technique for accurately analyzing gene expression. According to different methods, it can be divided into two categories: dye-based qPCR and probe-based qPCR, of which the dye-based method is more popular, convenient, and less costly. Dye-based qPCR enables indirect measurement of DNA amplification at each cycle of PCR by monitoring the fluorescence emitted by the dye bound to double-stranded DNA in real time. When the detected fluorescence signal significantly exceeds the background at a certain time point, the Ct value (Cq value) can be determined. The obtained Ct values can be used to assess the relative abundance of the gene of interest, or to obtain absolute numbers based on appropriate standard curve calculations.
Our product, HotStart™ Universal Blue 2X FAST Green qPCR Master Mix (with UDG), offers short extension times, superior specificity, robust amplification efficiency, ideal reproducibility and stability for quantifying target DNA or cDNA. It’s a 2X PreMix using a mutant hot-start fast Taq DNA polymerase that is more tolerant to SYBR Green I inhibition, as well as EDTA-treated blood and heparin-treated blood. The ideal Taq polymerase and suitable buffer guarantee superior specificity and high amplification speed. SYBR Green I in Mix interacts within the minor groove of double-stranded DNA, and emits green fluorescence, thus the amplification product can be indirectly quantified in real time by monitoring the fluorescence with an instrument.
Designed and developed for fast real-time PCR, this new product improves durability, specificity, and fast elongation speed, as well as improvements in signal-to-noise ratio (fluorescence), cyclic domain value (Cq), linearity, and sensitivity. The product also contains a Specific ROX Reference Dye, which is suitable for use with all qPCR instruments, eliminating the need to adjust the concentration of ROX on different instruments.
This product contains a visible blue dye to minimize aliquoting errors without affecting qPCR reactions or fluorescence signals. Additionally, thermolabile UDG and dUTP were included in the master mix to prevent carryover contamination between reactions. UDG pre-treatment of the reaction can ensure the accuracy of qPCR amplification results and reduce false positive results caused by aerosol contamination from previous reactions.
Dye-based qPCR has certain limitations, i.e., SYBR Green I can insert any double-stranded DNA, such as primer dimers or other non-specific products, resulting in fluorescence of non-specific products, to confirm product specificity, after amplification, melt curve analysis is necessary. In the analysis of the melting curve, a spike near the primer annealing temperature is an ideal result.
1. Using a mutated Fast Taq enzyme, the extension time can be reduced to as short as 20 seconds.
2. Using optimized components and proteins to increase the specificity of qPCR.
3. Containing dUTP and thermolabile UDG (K1109) to prevent aerosol contamination and false positives.
4. Containing special Specific ROX Reference Dye, suitable for all qPCR instruments without adjusting ROX concentrations.
5. Containing blue visible dye for easy loading.
