分子生物学常用技术PCR材料.pptVIP

* /chemistry/laureates/1993/mullis-lecture.html /chemistry/laureates/1993/mullis-autobio.html Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction. Scientific American. 262 (4) 56-65. * Good laboratory technique. Note that ethanol is not an effective way to clean you work area. DNA is not soluble in ethanol. We in fact use it to precipitate DNA. 10% bleach is probably the best approach as it will hydrolize as well as dissolve the DNA. Any consumer pump spray like 409 or Fantastic are less noxious and effective alternatives. Screw cap tubes prevent spray of your sample when you open the tube. Plugged tips prevent cross contamination. Calibration speaks for itself. Most people cannot pippette 2 uL reproducibly. It is difficult to tell when the volume is off because of a loose tip or worn pipettor. One can visualize 5uL in the tip and can usually tell when the volume is off. Using pooled master mixes for replicates tests the assay’s reproducibility and not the reproducibility of pipetting. Remember to vortex and spin down. * What is real time qPCR? It is fluorescence-based detection of amplification products through the use of a DNA-binding dye or probe chemistry. In a qPCR assay one measures the input level of a nucleic acid by determining the number of cycles required to reach a set level of product. This is in contrast to traditional PCR where end-point analysis is performed for product distinction. Shown in the left panel is a representation of a traditional PCR reaction. The reaction contains a certain amount of starting template to which the forward and reverse primers will anneal. After one round of amplification there is a doubling of product. After 30-40 cycles there will be an exponential accumulation of the specific product. After amplification, products are typically distinguished using endpoint, electrophoretic analysis. * This slide points out why traditional PCR is not a reliable method to quantify the sta