Question

17. Briefly describe the process of gel electrophoresis. Include an illustration. Be sure to label the positive and negative electrodes, the wells, and to indicate the direction that the DNA will move.

Answer 1

Ans) There are many types of gel electrophoresis namely Capillary electrophoresis,SDS PAGE electrophoresis,Native gel electrophoresis.

SDS PAGE electrophoresis is performed when the protein mixture is to be separated based on their size. Basically,the protein mixture gets negatively charged (by treatment with anionic detergent i.e SDS (sodium doedecyl sulfate) ) and now when the mixture or sample is placed in the electric field,it will migrate from cathode (negatively charged electrode) towards anode (positively charged electrode).The separation will be based on the charge and the relative mobility given by the formula

R_f =$\frac{ZE}{F}$  

where Z = charge on the molecule ,

F = Frictional resistance (when moving through the gel),

Rf is measured by

Distanceproteinbandmoves Distancedyefrontmoves

If Z < 0, then it moves towards anode (positively charged),

If Z>0,then it moves towards cathode (negatively charged),

If Z=0 , then no movement will occur (Proteins do not migrate at their isoelectric point.They are electrophoretically non-mobile at isoelectric point)

Here in this case, DNA is concerned and not the proteins. When DNA isolation is to be performed we use Agarose gel electrophoresis. The DNA is negatively charged due to the presence of phosphate groups and thus it will move from cathode (negatively charged) to anode (positively charged). The DNA sample of about 20$\mu$L is loaded into the wells and electrophoresis is performed on constant 100V, later after the gel has run approximately 3/4th the length , we remove the gel and stain it with Brilliant coomassie blue staining solution.Running buffer is Tris-glycine solution.