Ptc Genetics Lab Student Worksheet (Page 10 of 10) in pdf

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Appendix B - What is Gel Electrophoresis?

Looking at a sample of green dye, how can you know if it is really green? Could it be a mixture of blue and yellow dyes?

Electrophoresis is a technique used in many areas of science to analyze and separate samples by applying a constant

electric field. Biologists or forensic scientists can use this technology to separate mixtures of DNA or dyes into each

component based on size and electrical charge.

The gel in gel electrophoresis is essentially a matrix through which particles travel. Gels can be made from different

substances depending on what is being separated (DNA, RNA, proteins, etc.), but it should be both conductive and have

the ability to form a uniform matrix with appropriate pore sizes. The matrix is like a sieve or collander: if the holes are

too big or too small it wont work very well. One of the most commonly used and effective reagents for DNA separation

is agarose. Agarose gels are usually cast in a tray with molten (melted) agarose. A comb is placed while the agarose is

molten and then removed after the gel solidifies to create wells in which to load samples. A DNA stain is added to the gel

to enable visualization of the DNA, either before casting or after the run if DNA is going to be observed.

As an electric field is applied to the agarose gel, the particles in the wells will begin to move. The direction that particles

migrate depends on their charge. DNA has a negative charge, so it will be attracted to a positive electrode. Some dyes and

other particles have a positive charge and will thus migrate toward a negative electrode. The relative speed of migration

is determined mainly by the size of the particle but also by the strength of the particle’s charge. Like an obstacle course,

larger particles have more difficulty passing through the matrix with their bulk and do not travel very far, while shorter

and smaller ones can maneuver much more easily and therefore travel faster and farther.

Sometimes a particle with a bigger size migrates faster than a smaller particle. This can happen if the strength of the

charge of the larger particle is significantly stronger by comparison to the charge on the smaller particle. An example

of this phenomenon is the loading dye Orange G. This dye often runs faster than the smaller DNA fragments and other

relatively small particles because it is more negatively charged and has a stronger attraction to the electrode than the

smaller particles.

Both particle size and electrical charge can affect the results of gel electrophoresis experiments. In general however, gel

electrophoresis separates charged particles and fragments by size.

Ptc Genetics Lab Student Worksheet Page 10