qPCR优化说课.ppt

目标序列分析 二级结构分析 温度梯度优化实验条件 标准曲线验证 目标序列分析 二级结构分析 温度梯度优化实验条件 标准曲线验证 目标序列分析 二级结构分析 温度梯度优化实验条件 标准曲线验证 目标序列分析 二级结构分析 温度梯度优化实验条件 标准曲线验证 * Line is difficult to see. No amp * No amp * 费试剂！分两步做： Poor amp Optimization of the annealing temperature can be accomplished by running a cycling protocol with a temperature gradient across the thermal cycler block. With MJ real time instruments, you can identify the best annealing temperature in one step by using the dynamic thermal gradient feature. Shown here is the gradient calculator from the Opticon Monitor software that enables the user to program up to a 24oC range of annealing temperatures. * 回过头来看扩增结果 不同温度下，扩增效率不同，导致Cq值也不同，找Cq早的温度 （产率最高） Typical profile * 黑颜色是特定温度下的扩增曲线 Detailed look at resuts… Look at samples according to their annealing temperature and rate according to reproducibility and early Ct – these tow parameters are often combined. * Line is difficult to see. No amp * No amp * Poor amp * better * good * good * good * good * 如有范围，可选高一点 (特异性更高、更接近延伸温度、节约时间， 但不要选极端温度，以避免下次使用另一个试剂盒时候条件变化而结果不理想。 We have now proven that with our samples, reagents etc.. The optimal range for annealing for this particular primer set is between 55 and 60.7 degrees 前面说的都是优化 * This reviews setting up a dilution series. It is a SERIAL dilution. Occasionally a researcher will attempt to make all dilutions from the high concentration standard directly or will mix and match. It seems reasonable at first glance but extensive experience dictates that this will not produce optimal results. It is also worth pointing out that these should be made fresh every time you do the experiment. * 如果退火温度做得合适，引物浓度对Cq的影响会降到最低: 55C…Cq 差别很大 65C…及时引物浓度差8倍，这些线基本上是在一起的。 最佳退火温度的选择 最佳退火温度的选择 最佳退火温度的选择 最佳退火温度的选择 Optimal annealing temperature 最佳退火温度的选择 100nM each primer 引物浓度的优化 200nM each primer 引物浓度的优化 400nM each primer 引物浓度的优化 800nM each primer 引物浓度的优化 400nM primer at 60o 800nM primer at 60o 200nM primer at 60o 100nM primer at 60o