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Chapter 5 Molecular Tools for Studying Genes and Gene Activity
5.1 Molecular Separations Gel electrophoresis(凝胶电泳) staining with a fluorescent dye and the gel is examined under ultraviolet DNA is negatively charged cathode illumination The gel with slots DNA migrates toward anode agarose Figure 5.1 DNA gel electrophoresis. anode向 Mobility:Friction force In neutral pH buffer
Mobility: Friction force In neutral pH buffer 5.1 Molecular Separations Gel electrophoresis(凝胶电泳) The gel with slots DNA is negatively charged anode cathode staining with a fluorescent dye and the gel is examined under ultraviolet illumination agarose _ + anode
The mobilities of these fragments are plotted versus the log of their molecular weights (or number of base pairs) 2000p 1500 deviates strongly from linearity if the DNA is very large (mm Any unknown DNA can be electrophoresed in parallel with the standard fragments,and its size can be estimated if it falls within the range of the standards
logarithmic The mobilities of these fragments are plotted versus the log of their molecular weights (or number of base pairs) Any unknown DNA can be electrophoresed in parallel with the standard fragments , and its size can be estimated if it falls within the range of the standards deviates strongly from linearity if the DNA is very large
For the DNAs in the size ranges ~Mb(million base pairs,megabases) pulsed-field gel electrophoresis (脉冲凝胶电泳，PFGE) Instead of a constant current through the gel this method uses pulses of current with relatively long pulses in the forward direction and shorter pulses in the opposite,or Figure 5.3 Pulsed-field gel electrophoresis of yeast chromosomes. even sideways, direction·
For the DNAs in the size ranges ~Mb(million base pairs, megabases) pulsed-field gel electrophoresis (脉冲凝胶电泳,PFGE) Instead of a constant current through the gel， this method uses pulses of current， with relatively long pulses in the forward direction and shorter pulses in the opposite，or even sideways， direction．
polyacrylamide gel electrophoresis (PAGE,聚丙烯酰胺凝胶电泳) Sodium dodecy1 sulfate(SDS,十二烷基磺酸钠) M,(kD) Denaturing the subunits so they no longer 250 bind to one another. 160 105 The SDS has two added advantages: It coats all the polypeptides with negative charges so they all electrophorese toward 35 the anode(正极). It masks the natural charges of the subunits,so they electrophorese according to 15 0 their molecular masses and not by their native charges. Figure 5.4 SDS-polyacrylamide gel electrophoresis Pre-stained protein markers
Pre-stained protein markers polyacrylamide gel electrophoresis （PAGE,聚丙烯酰胺凝胶电泳) Sodium dodecyl sulfate(SDS,十二烷基磺酸钠) Denaturing the subunits so they no longer bind to one another. The SDS has two added advantages: ✓It coats all the polypeptides with negative charges，so they all electrophorese toward the anode(正极). ✓ It masks the natural charges of the subunits, so they electrophorese according to their molecular masses and not by their native charges