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It is helpful to include not only positive control samples for each allele, but also NTCs and heterozygotes. Note that positive samples typically have lower Cq values and higher RFU values than negative samples. The Allelic Discrimination tab allows the data to be displayed as Cq values or RFU values, depending on your preference. The autothresholds are most likely to be placed incorrectly when the variation among unknown samples differs from that among control samples. CFX Manager software typically positions the threshold automatically, but it may need to be adjusted in some cases. It is therefore essential that these thresholds be set appropriately for the alleles to be called correctly. The automatic calling of alleles is determined by the positioning of the threshold lines. Try to keep the amount of starting template similar among reactions. Store the PCR components properly especially take care to aliquot the probes in sufficiently small volumes to avoid multiple freeze-thaw cycles, and store the aliquots below –20☌. Prior to hot-start activation, use a qPCR supermix with good inhibition of polymerase activity. Cross- reactivity to nontarget alleles (for example, pseudogenes) can be problematic for assays developed using cloned products or other simplified targets. Optimize assay conditions using real samples whenever possible. Include NTC samples to detect template-independent reactions (for example, primer/probe dimers) and reoptimize assays if problems are detected. Use amplicons with a target length <300 bp to improve detection of sequence variants. Nonspecific amplification can occur due to the loss of hot-start enzyme activity, degraded reaction components, or too much starting template. Sometimes nonspecific amplification can occur, which may lead to false positive signals for one or more alleles. Use barrier tips to avoid contamination due to your pipet.Īllelic discrimination requires well-optimized assays. Load plate carefully and do not try to expel the residual volume from the pipet tip because this can cause bubbles which may spray droplets into adjacent wells. Run replicate samples (duplicates, triplicates, etc.) to help identify when loading contamination may have occurred. Do not work with contamination sources, such as post-PCR products, in the same area where PCR setup is done. Set up work space to avoid contamination. Include no-template control (NTC) samples to help detect global contamination, such as contamination of master mix components. Contamination during plate loading typically affects one or a few wells. Contamination in master mix components will show up in all samples.
Contamination can cause reactions that should otherwise fail to be positive for the allele. The ability to detect and amplify very small numbers of starting templates by PCR means that contamination control is always important.