QPCR原理.ppt

Mastercycler ep realplex - Software Data Analysis: End Point Detection Mastercycler ep realplex - Software Data Analysis: +/- Assays * * * * No Ventilation slots on the side: Space saving because the devises can be placed directly next to each other * A threshold is where the reaction curve enter and crossing into the exponential phase, anything below this line is considered as back ground while anything above the threshold is consider as the useful data. Ct value can be use as a way to plot a standard curve for quantitative analysis. * * * In relatvie, you not longer interested in adsolute, where you are actually looking at how many fold of expression of your gene of interest. The gene is up-regulated, because the sample gene of interested in move from the calibrator to sample house keeping gene is a normaliser, it should not be shifted. Under ideal condition, housekeeping should not be shifted. DDCt is the calcuation of the sample DCt minus calibrator DCt. We fold change not in adsolute value. * * * SYBR Green chemistry is an alternate method used to perform real-time PCR analysis. SYBR is a dye that binds the Minor Groove of double stranded DNA. The intensity of the flourescent emissions incrase as when it bind to any doble stranded DNA. The SYBR Green dye signal incrase as the amplicon number increases. * Temperature profile of the initial heating step with (blue line) and without (pink line) application of Impulse PCR * The another alternative method used to perform real-time PCR analysis is the Probe base system. It is a short DNA fraqment incorporate with a fluorecent dye, it is principle base on FRET, a principle that involve interaction of two florochrom on short distance. * * * * This mechine is fully upgradable, it is originally transform from a Ep gradient mastercycle, there’s two model here, one is the deluxe fast PCR silver block, another one is the standard aluminiam block these two thermol module can upgrade by adding an optical module and be