Probe qPCR MasterMix
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Cat. No.: PQM-5 (for 5 ml)
Cat. No.: PQM-25 (for 25 ml)
Cat. No.: PQM-250 (for 250 ml)
Description
Probe qPCR MasterMix uses DNA template and qPCR primers to carry out fluorescence quantitative PCR. It is easy to operate, minimizes human errors, effectively reduces the risk of contamination, saves the operation time of the PCR experiment, and has large detection throughput.
Detection Principle
Probe-based qPCR uses fluorescent and quencher-labeled DNA probes to target the sequence which will be amplified by PCR. Normally, quenching groups on the probe result in quenching of fluorescent groups due to the fluorescence resonance energy transfer (FRET) in space. When the target sequence is amplified by PCR reaction, both primers and probes are annealed to the target gene. With the extension of primers, the 5' to 3' exonuclease activity of Taq polymerase will cause the probe bound to the target sequence to be degraded gradually from the 5' end. After the fluorescent group and quenching group of the probe are cleaved by Taq polymerase, the quenching group disappears, and the fluorescent group can be normally excited by the excitation light to produce fluorescence. After each PCR cycle, more fluorescent groups are released, and the fluorescence intensity is proportional to the number of newly synthesized target fragments, thus quantitative detection can be achieved. Probes are usually a fragment of linear DNA specific to the target sequence, labeled with fluorescent groups such as FAM or HEX at the 5' end and fluorescent quenching groups such as BHQ1, TAMRA, or MGB at the 3' end.
Features
- High specificity and sensitivity: Specificity is not only dependent on PCR primers but also specific binding and degradation of probes and target genes to generate fluorescent signals. The detection sensitivity and specificity are usually significantly higher than those of the methods using fluorescent dyes such as SYBR Green.
- Multiple detections: In a single reaction, different genes correspond to different probes and different probes correspond to different fluorescent markers, which can be used for multiple fluorescent quantitative PCR detection. Probe qPCR MasterMix can be used for the detection of 2-3 genes at the same time after optimization of primers and probes.
- Inhibition of nonspecific amplification: The polymerase in the mastermix has hot-start property, which can effectively avoid nonspecific amplification caused by nonspecific annealing of primer and template DNA or primer dimer at low temperature.
Storage
The minimum shelf life is a year at -20°C with light-free. Avoid repeated freeze-thaw cycles.
Instruction: Protocol
Related: SYBR Green qPCR Mastermix, Probe One-Step RT-qPCR MasterMix, PrimeTaq™ Probe qPCR MasterMix
Only for research and not intended for treatment of humans or animals
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