LightCycler480荧光定量PCR仪散热元件Therma-Base.ppt

* Gene Scanning Analysis – Workflow Create a Gene Scanning Analysis Select the subset of samples you want to analyze… * Gene Scanning Analysis – Workflow Normalization Fluorescence values acquired during High Resolution Melting will vary in magnitude, e.g., due to differences in the starting amount of template in each sample. This variability can mask differences between genotypes. Therefore the first step in the analysis process is to normalize the data. Raw melting curve data are normalized by setting the pre-melt (initial fluorescence) and post-melt (final fluorescence) signals of all samples to uniform values. Pre-melt signals are uniformly set to a relative value of 100%, while post-melt signals are set to a relative value of 0%. Normalizing the initial and final fluorescence in all samples helps interpretation and analysis of the data. The upper graph, the Melting Curves graph, contains two pairs of vertical sliders. The green sliders correspond to Pre-melt Low and High temperatures, the blue sliders to Post-melt Low and High temperatures. The grey area between the sliders indicates the area used for normalization. The software automatically places the temperature sliders in a suitable region for normalization and displays the normalized data in the lower graph, the Normalized Melting Curves graph. Examine the upper graph to make sure that the Pre-melt Temperature Range lies in an area where the background fluorescence of all the samples is dropping consistently but no temperature transitions have occurred, and that the Post-melt Temperature Range is placed in an area where melting is complete for all samples. If necessary, adjust the temperature settings by either dragging the vertical sliders on the upper graph or by changing the values in the Slider Settings fields. For most purposes, the recommended temperature interval between each set of vertical sliders is about one degree. * Gene Scanning Analysis – Workflow Temperature Shift Eliminating the tempe