CFX96 实时荧光定量PCR培训教材PPT要点.ppt

EvaGreen 饱和荧光染料 Just import CFX Melt Data Normalize curves Precision Melt - Genotyping * To these profiles, We set a benchmark, the Threshold line (T). This is an arbitrary line that can vary quite a bit. It does have one restriction, it must be set during the exponentially growth phase of the amplifications. From this threshold, we collect CT values. These values simply represent the number of cycles the sample required to reach the amount of product (fluorescence) marked by the Threshold line. * If you know the amount of target DNA present in the samples at the start… These CT’s can be plotted as a function of starting quantity and generate a standard curve. * Any unknowns that want to be quantified are run concurrently with our standards. The CT of the unknown is collected……. * …. And plotted against the standard curve. This gives us an accurate determination of the number of copies of our target DNA in our unknowns. A simple analogy: QPCR is similar to running a protein assay such as a Bradford or a Lowry. The difference is that instead of using a genetic standard such as BSA, QPCR requires the gene itself to be used as a standard. What we are actually doing is determining the efficiency of our assay in order to determine relative to a standard, what is I the unknowns. * 荧光曲线之间的间距由等式“2n = 稀释倍数”决定，这里 n 是阈值线上扩增曲线之间的循环数（或称为CT的差异） 浓度增加1倍，CT值减小1个单位 ?浓度增加10倍，CT值减小3.3个单位 * SYBR Green I dye has an increased fluorescent intensity when bound to double stranded DNA. * When sample is denatured, signal drops. When more double stranded DNA is generated, the signal increases proportionally with the amount of dsDNA present. * When running a SYBR assay it is essential to run a melt curve to confirm that only one product is being amplified. The dye has no way of discriminating between the amplicons we want from those we don’t. Due to an additive effect of the signal, any samples displaying multiple products cannot be accurately quantified. * How does melt curve wo