What are the advantages of SYBR Green?
The main advantage of using SYBR® Green for qPCR is that there is no requirement for the incorporation of a fluorescent reporter system into the primer design or the synthesis of fluorescently labeled probes or beacons that are specific only for a target sequence.
Why is TaqMan more specific?
This method is relatively cost benefit and easy to use. Specificity is the most important concern with the usage of any of these non-specific dsDNA-binding dyes. Non-specific products reflected in dissociation curve of the amplified product as non-specific peaks.
What is TaqMan qPCR?
TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection.
Does TaqMan use fret?
5′ nuclease assay for PCR monitoring (TaqMan probes) The 5′ nuclease assay was developed to allow the real-time monitoring of the reaction. In its initial format, it did not use FRET but relied on radioactive labeling instead (35).
Why are primer dimers bad?
Mostly primer dimers are seen due to high concentration of the primer in the PCR reaction mixture. As a result, the DNA polymerase amplifies the Primer dimer , leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification.
How do you avoid primer dimers in qPCR?
i suggest one (or more) of the following solutions:
- increase the annealing temperature.
- increase time\ temperature of template denaturation.
- decrease primers concentration(10 pmol will be OK)
- use a PCR enhancer such as DMSO.
- Check out your template.
- use high quality Tag.