K1170-25 | HotStart™ Universal 2X Green qPCR Master Mix Clinisciences

Description

Quantitative PCR (qPCR, also called Real-time PCR) is a popular technology for precise analysis of gene expression. It can be classified into two categories according to different methods, dye-based and probe-based, in which, dye-based method is more popular, convenient and less costly. Dye-based qPCR monitors real-time fluorescence of the dye binding to the double-stranded DNA to measure DNA amplification indirectly during each cycle. At a point where the fluorescence signal is distinctly detected over the background, Ct value (Cq value) can be determined. The obtained Ct values can be used to evaluate relative target abundance or calculate absolute target quantities in reference to an appropriate standard curve.

HotStart™ Universal 2X Green qPCR Master Mix provides superior specificity, robust amplification efficiency, ideal reproducibility and stability in quantifying target DNA or cDNA. It’s a 2X premix, taking advantage of a hot-start Taq polymerase combined with the antibody. Ideal Taq polymerase and suitable buffer will guarantee preferable specificity and high amplification speed. Green I in the mix which is a DNA intercalator will emit fluorescence when bounds to the double-stranded DNA amplified in each cycle and monitoring the fluorescence by the instrument allows for the indirect quantification of amplification products at real time. This reagent has special Specific ROX Reference Dye which is suitable for all qPCR instruments. Instead of adjusting the concentration of the ROX on different instruments, you can simply add primers and templates to amplify when formulating the reaction system.

However, dye-based qPCR has some limitations. Green I can intercalate into any double-stranded DNA, such as primer dimer or other undesired products, causing nonspecific products to emit fluorescence. To confirm the specificity of the product, following amplification, performing a melt curve analysis is necessary. A sharp peak around the annealing temperature in the melt curve analysis is ideal.

Product Citation

1. Xiaotian He, et al. "Tumor-derived apoptotic extracellular vesicle-mediated intercellular communication promotes metastasis and stemness of lung adenocarcinoma." Bioact Mater. 2024 Mar 6:36:238-255. PMID: 38481566
2. Zhenzhen Chu, Baohuan Zhang, et al. "A DNA/RNA heteroduplex oligonucleotide coupling asparagine depletion restricts FGFR2 fusion-driven intrahepatic cholangiocarcinoma." Mol Ther Nucleic Acids. 2023 Oct 5:34:102047. PMID: 37869260
3. Hancheng Fan, Jiaqiang Wu, et al. "Endoplasmic reticulum stress negatively regulates intestinal stem cells mediated by activation of GRP78/ATF6/CHOP signal." Research Square. 22 Aug, 2023.

Quality Control

Protocol

Components and Storage

Components	5mL 1000 rxn with 10μL reaction 500 rxn with 20μL reaction 200 rxn with 50μL reaction	25mL 5000 rxn with 10μL reaction 2500 rxn with 20μL reaction 1000 rxn with 50μL reaction	50mL 10000 rxn with 10μL reaction 5000 rxn with 20μL reaction 2000 rxn with 50μL reaction
HotStart™ Universal 2X Green qPCR Master Mix	1 mL x 5	1 mL x 25	5 mL x 10
Store at -20°C.

HotStart™ Universal 2X Green qPCR Master Mix

Referentie K1170-25

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Description

Product Citation

Quality Control

Components and Storage

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