/Gel electrophoresis protocol pdf

Gel electrophoresis protocol pdf

Standard gel electrophoresis techniques for separation of DNA molecules provided huge advantages for molecular biology research. However, it was unable to separate very large molecules of DNA effectively. 20 kb migrating through a gel will essentially move together in a size-gel electrophoresis protocol pdf manner.

The pulse times are equal for each direction resulting in a net forward migration of the DNA. This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel. 50 kb where all large fragments will run at the same rate, and appear in a gel as a single large diffuse band. However, with periodic changing of field direction, the various lengths of DNA react to the change at differing rates. That is, larger pieces of DNA will be slower to realign their charge when field direction is changed, while smaller pieces will be quicker. Over the course of time with the consistent changing of directions, each band will begin to separate more and more even at very large lengths.

Thus separation of very large DNA pieces using PFGE is made possible. PFGE may be used for genotyping or genetic fingerprinting. It is commonly considered a gold standard in epidemiological studies of pathogenic organisms. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis”.

This page was last edited on 8 February 2018, at 00:26. The DNA size marker is a commercial 1 kbp ladder. The position of the wells and direction of DNA migration is noted. Agarose gel is easy to cast, has relatively fewer charged groups, and is particularly suitable for separating DNA of size range most often encountered in laboratories, which accounts for the popularity of its use. The separated DNA may be viewed with stain, most commonly under UV light, and the DNA fragments can be extracted from the gel with relative ease. Most agarose gels used are between 0.