Real-Time PCR实验.ppt

* 扩增片段越短，扩增效率越高，扩增片段越长，R值越好。 * There are many variables that affect precision, some a function of the instrument and chemistry, but also the operator and set-up tools. For quantitative work, precision is essential. Click 1: Pipettors, perhaps not surprisingly, are often a weak link. Use the one pipettor where possible to remove pipettor-to-pipettor inaccuracy. Also use quality tips. Low quality tips may not always fit snugly onto the pipettor barrel and thus draw-up variable sample quantities. Some cheap colored tips are known to leach dye into the samples—this can be disastrous for a fluorescent reaction! Click 2: Dispensing reagents comes down to operator expertise. Good practice is essential, and some of the basics are listed here. The fact is, if you make pipetting errors, this technology will highlight them. From experience we know that operators will improve their results over time and it all comes down to practice and the unforgiving feedback the process provides. Great precision into the reaction (as reaction components) means great precision out (as reported results), but the reverse is also true. From an instrumentation point of view, you want an instrument that controls variables as much as possible. There thermal, optical and instrument variables involved. Click 3: Thermal precision is the measure of how much variation occurs between each sample. Thermal accuracy is different, it’s a measure of how close the set temperature is to the absolutely correct temperature. This is dependant on proper instrument calibration, which we’ll address separately. Click 4: Equilibration time is also important. In the RG, all tubes change temperature at the identical rate. In other systems the tube position affects the rate at which it heats or cools—some tubes are nearer the heating or cooling elements, for example. Of course, if you have to wait for 10-20 seconds for temperature to equilibrate you cannot collect meaningful data until then. Click 5: The optical s