Developing multiplex Real-Time PCR assays can be difficult and time-consuming. As the reaction complexity increases, significant optimization may be required to generate reliable data. It can be a challenge to develop multiplex assays that amplify all targets with equal efficiency. [...]
The specificity of any Real-Time PCR assay, whether TaqMan probe or SYBR Green I, is determined by the quality of the assay design. Non-specific amplification can occur for both SYBR Green I or TaqMan probe methods if the assay design [...]
There are two major detection chemistries used for Real-Time PCR: enzymatic and hydrolysis (TaqMan) probe-based chemistries, and DNA-binding SYBR Green I dye-based chemistry. Additional detection chemistries include Molecular Beacons, Scorpions probes and LUX primers.
I am currently running traditional PCR; can I use the same template, primers, and reagents to run Real-Time PCR?
In some cases it is possible to convert existing traditional PCR assays into Real-Time PCR assays, with a few considerations around primer design and master mix. Primer design is one of the first considerations for converting a traditional PCR assay. [...]
xxpress® is an open platform that supports all current Real-Time PCR detection chemistries including DNA binding dyes, hydrolysis probes, hybridisation probes, as well as other detection chemistries such as molecular beacons and Scorpion primers.
Yes, the xxpress® system is compatible with the following dyes or their generic equivalents: FAM/SYBR, TAMRA, Texas Red, Cy5, Cy5.5. Channel Excitation (nm) Emission (nm) Example Fluorophores Detected 1 470 513–531 SYBR Green I, FAM 2 530 569-588 TAMRA [...]
In Real-Time RT-qPCR, genomic DNA can potentially be co-amplified during the PCR reaction, contaminating the sample and leading to erroneous results. To determine if an RNA sample is contaminated with genomic DNA it is important to include a no-reverse transcriptase [...]