罗氏荧光定量PCR.PPT

ET: PCR Efficiency for traget molecules ER: PCR Efficiency for reference molecules Cp: Amplification cycle at crossing point; CT: Threshold Cycle S: Sample C: Calibrator The error generated when assuming the efficiency of a PCR reaction is = 2 but the actual efficiency is = 1.95. After 35 cycles of PCR, the quantified amount of sample material will have 142% error, a greater than 2-fold difference than the actual amount. Further down in this table, if you look at a PCR efficiency of 1.8, the error at 35 cycles is nearly 4000%. * RT时做2-3个平行反应，每个样本采用相同的RNA浓度 When a multiwell plate is inserted, it is carefully positioned on the multiwell plate mount, which provides a perfect fit with the plate. Heating and cooling is done using Peltier elements. These consist of semiconductors sandwiched between two ceramic plates, and work as electronic heat pumps. Their function can be switched to cooling by reversing the current. Underneath these Peltier elements, we have something quite special that plays a very important role for optimized heat transport to and from the plate: it is called a Therma-Base. The Therma-Base is a very thin sealed vacuum vessel that transfers heat by the evaporation and condensation of an internal working fluid. When the fluid condenses, it gives up its latent heat to the surrounding air, or, even better, in this case, to a closely connected cooling element or heat sink. The heat sink is a cooling element made of metal; therefore, it has a high thermal conductivity. Fins positioned on the inside ensure that the inner surface area is maximized, and a fan facilitates absorption of heat by the surrounding cooler air. Using the heat sink in combination with the Therma-Base improves heat dissipation and reduces the time required for the cooling steps within the cycling protocol. The block cycler comes with a heated cover containing pinholes; these are necessary to allow the central portion of the fluorescent signal to be