HotStart™ 2X Probe 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 principles, it can be divided into two categories: dye-based method and probe-based method. Commonly used probes include Taqman probes, molecular beacons, dual hybridization probes, MGB probes, etc. Among them, the TaqMan probe is the most classic and widely used. Taqman probes can be hybridized specifically to the target sequence, with a 5' end labeled fluorescent reporter and a 3' end labeled quencher. When the probe is intact, the fluorescent reporter on the probe is quenched by the quencher due to fluorescence resonance energy transfer (FRET). When the primer-mediated extension reaches the probe position, Taq enzyme with 5' → 3' exonuclease activity hydrolyzes the probe bound to the target sequence, lead to the separation of the fluorescent reporter at the 5' end and the quencher at the 3' end, apart more than 10 nm, beyond the distance of FRET, then the fluorescent reporter emits fluorescence with appropriate excitation light. With each PCR cycle, more fluorophores are released, and the fluorescence intensity is proportional to the number of newly synthesized fragments, enabling quantitative detection. Commonly used fluorescent reporters include FAM, HEX, TET, JOE, NED, Cy3, TAMRA, Cy5, Cal Fluor Gold540, Cal Fluor Orange560, etc., quenchers BHQ1, BHQ2, BHQ3, TAMRA, Dabcyl, NFQ-MGB, etc. When designing probes, pay attention to different models of the qPCR machine (different qPCR machines have different fluorescent channels), various abundance of target genes, and the types of experiments.
The HotStart™ 2X Probe qPCR Master Mix is a high-quality master mix used in probe-based real-time PCR. It’s a 2X PreMix using a mutant hot-start Taq DNA polymerase with improved template affinity, resistance to PCR inhibitory components such as blood, serum IgG, humic acid, etc. This product is particularly suitable for quantitative or qualitative detection of low-abundance or high-specificity genes of interest.
This product is a 2X master mix that requires only additional primers, probes, and templates, which is convenient to use and compatible with fast procedures, that means shortened time to generate the result, and enables accurate, highly repeatable quantification of target gene over a wide dynamic range. This product can be used for multiplex qPCR and supports up to quadruplex qPCR.
| 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 Probe 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 all components at -20°C and protect from light for 12-24 months. Avoid repeated freeze/thaw cycles as possible. | |||
