HotStart™ 2X FAST Green qPCR Master Mix
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™ 2X FAST Green qPCR Master Mix, 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 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. 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.
Dye-based qPCR has certain limitations, i.e., 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.
| Components | 5 mL 1000 rxn with 10 μL reaction 500 rxn with 20 μL reaction 200 rxn with 50 μL reaction |
25 mL 5000 rxn with 10 μL reaction 2500 rxn with 20 μL reaction 1000 rxn with 50 μL reaction |
50 mL 10000 rxn with 10 μL reaction 5000 rxn with 20 μL reaction 2000 rxn with 50 μL reaction |
| HotStart™ 2X FAST Green qPCR Master Mix | 1 mL X 5 | 1 mL X 25 | 5 mL X 10 | 50X ROX Reference Dye (low concentration) | 0.2 mL | 1 mL | 1 mL X 2 | 50X ROX Reference Dye (high concentration) | 0.2 mL | 1 mL | 1 mL X 2 |
Store the components away from light at -20°C for 12-24 months. | |||