Electrofocusing Gels: Another variation of gel electrophoresis is to pour a gel that purposely has a pH gradient from one end to the other. As the protein travels through this pH gradient, its various ionizable groups either pick up or lose protons. Eventually, it will find a pH where its charge is zero and it will get stuck at that point.
DNA Agarose Gels: A simple way of separating large fragments of DNA from one another by size is to use an agarose gel. Agarose is another type of matrix used for many purposes (such as the support for the growth of bacteria on plates). DNA does not need a detergent, since it is already has a large under of negative phosphate groups evenly spaced. Thus, as with SDS-PAGE, the charge to mass ratio is constant. In addition, like SDS-PAGE the separation results from the matrix itself. The range of size sensitivity can be varied by changing the density of agarose.
DNA Denaturing Polyacrylamide Gels: To look at smaller DNA molecules with much higher resolution, people generally denature the DNA via heat and run it through a thin polyacrylamide gel that is also kept near the denaturing temperature. These gels usually contain additional denaturing compounds such as Urea. Two pieces of DNA that differ in size by 1base can be distinguished from each other this way.
Capillary Electrophoresis: This is an automated analytical technique that separates species by applying voltage across buffer filled capillaries. It is generally used for separating ions, which move at different speeds when the voltage is applied depending on their size and charge. The solutes are seen as peaks as they pass through the detector and the area of each peak is proportional to their concentration, which allows quantitative determinations. Analysis includes purity determination, assays, and trace level determinations. Analysis times are in the region of 1-30 minutes depending on the complexity of the separation.
Immunoelectrophoresis: There are two major types of electrophoresis: protein electrophoresis and immunoelectrophoresis. Immunoelectrophoresis is used to assess the blood levels of specific types of proteins called immunoglobulins. In immunoelectrophoresis, a gel is prepared with alternating wells and slots cut into it. The Ag mixture (usually a serum sample) is placed in the wells and electrophoresis is performed to separate the proteins in the sample. Then an Ab (or mixture of Abs) is added to the slots in the gel. The separated Ags and the Abs migrate toward one another and form precipitin arcs at the region of optimal concentrations.
Pulsed-Field Gel Electrophoresis: Extremely large molecules of DNA (more than 106 base pairs) are effectively separated in agarose gels using PFGE. This technique employs two or more electrodes, placed orthogonally with respect to the gel, that receive short alternating pulses of current. PFGE allows whole chromosomes and large portions of chromosomes to be analyzed.
Paper Electrophoresis: In this technique, a strip of paper is kept moist with buffer to make it electrically conductive; ends are dipped into buffer solutions containing electrodes across which an electrical potential is applied. A high voltage must be used here otherwise samples diffuse too rapidly. Paper electrophoresis is used primarily for separation of small molecules.
Observation
Results and DiscussionAgarose gel: This is a linear polymer of L&D galactoses purified from agar. Its gel is made by dissolving agarose powder in hot buffer. When this solution cools it becomes a gel having many large pores. For this reason, agarose is frequently used in DNA electrophoresis.
Buffer: Buffer is a mixture of chemicals that holds the pH constant during electrophoresis. There are three types of buffers; tris-acetate EDTA, tris-borate EDTA, tris-phosphate EDTA. In this experiment we used tris acetate EDTA(TAE). TAE is a basic mixture of pH 8.
Ethylene Diamine Tetra Aceticacid (EDTA): EDTA is used to hold metal ions in order to protect the DNA from being digested by nucleases and DNAases, as these enzymes use metal ions as cofactors to function.
Ethidium Bromide (EtBr): This is a highly toxic and carcinogenic fluorescent dye used to visualize DNA. After the experiment is complete if the gel is exposed to UV ethidium bromide allows the DNA to be seen as flashing bands. There are two ways of staining the gel with EtBr:
- Incorporating EtBr into gel while preparing
- Immersing gel into EtBr after the experiment
Bromophenol Blue(loading dye): The dye has three functions in the experiment.
- Increases density of the sample so that when the sample is loaded into wells it does not go up. The sample has to go through the gel in order to separate. If it does not and goes up, the experiment will be useless.
- Gives colour to the sample so that it enables loading of the samples into wells, done easier.
- Helps to decide when to stop electrophoresis.
Factors Affecting the Migration:
- Size of DNA: the size and migration rate are inversely proportional
- Charge of DNA: charge is determined primarily by the sugar phosphate backbone but in this experiment charges of different DNAs were nearly equal
- Concentration of agarose: 0.8%agarose gel used for 8000-10000 bp molecules
1% “ “ “ “ 4000-8000 “ “
1.5% “ “ “ “ 2000-4000 “ “
2% “ “ “ “ 1000-3000 “ “
- Voltage applied: voltage and migration rate are directly proportional
- Conformation of DNA: there are three types of plasmid DNA: supercoiled, opencircular and linear. Of these three supercoiled moves the fastest.
In our experiment, migration was mostly determined by size and shape. Actually the force on the molecules is due to the electric field acting on the charge of the molecule and is given by F=E*q. Obviously, the greater the charge on the molecule, the greater the force. But it is less obvious that molecules with a larger mass will move more slowly. This comes about because of the frictional forces that slow a molecule travelling through a solution down depends on the size of the molecule. The greater the mass of the molecule, the greater the size and therefore a greater frictional force on the molecule moving. Frictional force on the molecule also depends on the shape of the molecule in our case. Spherical molecules (supercoiled and opencircular) go easily through pores of the agarose gel and friction on them is less, whereas on rodlike (linear) molecules the friction force is more as they can not move easily through the pores.
