Gel Electrophoresis Research http://www.dnalc.org/resources/animations/gelelectrophoresis.

html Gel Electrophoresis In the early days of DNA manipulation, DNA fragments were laboriously separated by gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This process uses electricity to separate DNA fragments by size as they migrate through a gel matrix. Gel Electrophoresis can be used to separate DNA fragments. Electrophoresis uses an electric current to separate different-sized molecules. In a porous , sponge-like matrix. Smaller molecules move more easily through the gel pores than larger molecules. While at Cold Spring Harbor Laboratory, Phi Sharp, Joe Sambrook, and Bill Sugden developed the DNA electrophoresis technique using an agarose gel, made from highly purified seaweed. This could be used to separate DNA molecules ranging from several hundred nucleotides in length to over 10,000 nucleotides. The gel is submersed in a tank filled with a salt solution that conducts electricity. Using a pipette , DNA samples are loaded into slots made in the agarose gel. The DNA samples are colourless, but researchers add a blue tracking dye. This makes it easier to load the samples , and visually track the DNA migration through the gel. The phosphate groups in the DNA backbone carry negatively-charged oxygens giving a DNA molecule an overall negative charge. In an electric current, the negatively-charged DNA moves toward the positive pole of the electrophoresis chamber. The DNA molecules move through the gel by reputation a reptile like snaking action through the pores of the agarose matrix. Smaller DNA fragments migrate faster and further over a given period of time than do larger fragments. This is how DNA fragments can be separated by size in an agarose gel. Sharp, Sambrook and Sugden introduced the use of the fluorescent dye, ethidium bromide, to stain DNA. Ethidium bromide binds tightly to the DNA double helix, and glows when illuminated with ultraviolet light. This lets researchers see where the separated DNA fragments end up. A photo of the gel can be taken for later analysis. The size of any DNA fragment can be determined by comparing it to markers DNA fragments of known sizes. A map of restriction enzyme sites can be generated by cutting a piece of DNA with different combinations of restriction enzymes. http://www.life.illinois.edu/molbio/geldigest/electro.html

Experiment 2: Gel Electrophoresis of DNA

What is Electrophoresis? Electrophoresis is a technique used in the laboratory that results in the separation of charged molecules. In this CyberLab we are separating molecules of DNA that we got from Restriction Digestion. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose. Click on the image to the left to see a larger image of typical equipment used in electrophoresis.

What is a Gel? You may be wondering what exactly a gel is, and what it has to do with agarose. Let's find out by "making" a gel. Purified agarose is in powdered form, and is insoluble in water (or buffer) at room temperature. But it dissolves in boiling water. When it starts to cool, it undergoes what is known as polymerization. Rather than staying dissolved in the water or coming out of solution, the sugar polymers crosslink with each other, causing the solution to "gel" into a semi-solid matrix much like "Jello" only more firm. The more agarose is dissolved in the boiling water, the firmer the gel will be. While the solution is still hot, we pour it into a mold called a "casting tray" so it will assume the shape we want as it polymerizes (otherwise it will just solidify in the bottom of the flask wasting the expensive agarose). Look through the sequence of images below to learn how to prepare a gel.

How are Gels Loaded and Run? Imagine you are a DNA molecule. If you were inside an agarose gel, your environment would resemble a very dense spider web. If you are a small fragment, you could easily crawl through the spaces in between the webs (they are too tough for you to just pull out of the way). But as you increase in length, it gets harder and harder for you to fit through the spaces. If it were a race between you and another DNA molecule, who would win? Do you think the same would hold true for any charged molecule? Now it's time to take the DNA we digested in Experiment 1 and load it on the gel we just prepared. So again, follow through the pictures below to load and run our gel. http://faculty.plattsburgh.edu/donald.slish/electrophoresis.html Agarose Gel Electrophoresis Introduction: Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs. Electrophoresis: The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move toward the positive pole:The rate at which the DNA will move toward the positive pole is slowed by making the DNA move through an agarose gel. This is a buffer solution (which maintains the proper pH and salt concentration) with 0.75% to 2.0% agarose added. The agarose forms a porous lattice in the buffer solution and the DNA must slip through the holes in the lattice in order to move toward the positive pole. This slows the molecule down. Larger molecules will be slowed down more than smaller molecules, since the smaller molecules can fit through the holes easier. As a result, a mixture of large and small fragments of DNA that has been run through an agarose gel will be separated by size. This is a graphic representation of an agarose gel made by "running" DNA molecular weight markers, an isolated plasmid, and the same plasmid after linearization with a restriction enzyme:

These gels are visualized on a U.V. trans-illuminator by staining the DNA with a fluorescent dye (ethidium bromide). The DNA molecular weight marker is a set of DNA fragments of known molecular sizes that are used as a standard to determine the sizes of your unknown fragments. If you click on the figure you will see a short movie that simulates the movement of the DNA bands through the gel. When looking at the video, note that bands of a low molecular weight move very quickly through the gel while high molecular weight bands move very slowly. Figure 2

Interpretation: Much information can be derived from this gel. As you read the text below, refer back to figure 2. 1.) By looking at the migration of the DNA molecular weight standards, you can tell that the migration of DNA through an agarose gel is not linear with respect to size. If you graphed the distance traveled vs. the molecular weight of the fragment, you would see that there is a logarithmic relationship (i.e. small fragments travel much faster than large fragments). 2.) You can see that there is a big difference between the way a plasmid as isolated from the alkaline lysis prep will run vs. this same plasmid after it is cut with a restriction enzyme and linearized. This is because the plasmid will be found in many different supercoiled forms in the bacteria. When you isolate plasmid from a bacterial culture, you isolate all the different supercoiled forms of the plasmid, and each will migrate differently on the gel, giving you three major bands and many minor bands. When this mixture of supercoiled plasmids is cut with a restriction enzyme, the different forms linearize and unwind. As a result they all become identical and run at the same rate, and you see only one band on the gel. 3.) The molecular size of an unknown piece of DNA can be estimated by comparison of the distance that it travels with that of the molecular weight standards. This is only true for linear DNA. None of the supercoiled forms will migrate at a rate relative to linear DNA, which means that you can't use the DNA markers to estimate the molecular weight of a circular DNA molecule. To estimate the molecular weight of a plasmid, you must first linearize it. By looking at the gel above, the molecular size of the plasmid can be estimated at approximately 3.0 kilobases (kb). A more accurate estimate can be found by graphing the molecular weight of the standards (in base pairs) vs. the distance traveled on semi-log paper and using this graph to determine the molecular weight of the unknown. You will do this at the end of this experiment. Molecular size is the most important information derived from the agarose gel and the usual reason for running a gel.

In this experiment, you will linearize the plasmid that you isolated last week with a restriction enzyme. Then you will run this linearized plasmid on an agarose gel with the uncut version and a DNA marker to determine the size of your plasmid + insert, which will give you an estimate of the size of your insert.

Procedure: 1.) Put together the following reaction mixture for the restriction digestion: 14.5 ul water 2.0 ul 10X Rest. Enzyme buffer 3.0 ul plasmid DNA solution (from last week) 0.5 ul Restriction Enzyme (eg., HindIII) 20.0 ul Total

Add the enzyme last, and always keep it on ice. The enzyme you will use will depend on the plasmid that you have, and will be told to you during class. 0.5 ul can't be measured with your pipetman. You must estimate it by the way it will look in the pipet tip (instruction will be given in class). Be sure to use a clean tip when taking the enzyme out of the tube. Put this reaction at 37oC for 45 minutes. 2.) When the digestion is complete, prepare to load the gel. In a new tube, place 17.0 ul of H 2O and 3.0 ul of uncut plasmid DNA. Add 2.0 ul dye to each of the three sample tubes (DNA markers, uncut plasmid, and digested plasmid). Load 20.0 ul of DNA marker in to one well of the gel. Do this by sucking the solution into the pipet tip, placing the tip in the top of the well, and gently expelling the liquid into the well. The dye buffer in the DNA marker and samples contains glycerol which makes it more dense than H 2O. This will cause the liquid to sink to the bottom of the well. Load 20.0 ul of the uncut plasmid and the restriction digestion. 3.) Turn on the power supply and electrophorese the samples at 110 V (Warning- be careful of the high voltage or you will be set down on your butt dramatically.) Electrophorese the samples until the dark blue dye is about 2 cm from the bottom of the gel 4.) Stain the gel by incubating it for 8 min in an ethidium bromide solution. http://www.google.com/url?sa=t&rct=j&q=gel+electrophoresis&source=web&cd=14&ved=0CI0BEBYw DQ&url=http%3A%2F%2Fwww.colorado.edu%2FOutreach%2FBSI%2Fpdfs%2Fgel_electrophoresis.pdf &ei=3FqXT9HXEY2ciAfDwZ2nCg&usg=AFQjCNGO5inZm5FZkslaTOyJaMig-eZUEw Biological Sciences Initiative Gel Electrophoresis Introduction DNA in a test tube all looks the same. It is impossible to tell the size of the DNA, or what the DNA encodes just by looking at the tube. Gel electrophoresis is one of the techniques scientists use to look at the DNA they have. This technique separates DNA molecules by size. How gel electrophoresis works First a gel is prepared. Gels are made of agarose, a seaweed extract similar to gelatin. The finished gel has a consistency similar to very firm jello. This consistency offers resistance to the pieces of DNA as they try to move through the gel. The gel is prepared with wells at one end so that DNA samples can be loaded into the gel. Once the DNA samples are loaded onto the gel, an electric current is applied to the gel. DNA is negatively charged due to all the phosphate groups in the backbone of DNA. Thus, DNA will move towards the positive electrode. As the pieces of DNA move through the gel, they will meet with resistance. Larger pieces of DNA will have more difficulty moving through the gel than smaller fragments. Thus, larger fragments will move slower than smaller fragments. This allows separation of all different sizes of DNA fragments. Uses of Gel Electrophoresis Gel electrophoresis is used to provide genetic information in a wide range of data fields. Human DNA can be analyzed to provide evidence in criminal cases, to diagnose genetic diseases, and to solve paternity cases. Samples can be obtained from any DNA-containing tissue or body fluid, including cheek cells, blood, skin, hair, and semen. In many analyses, polymerase chain reaction (PCR) is used to amplify specific

regions of DNA that are knownto vary among individuals. A persons DNA fingerprint or DNA profile is constructed by using gel electrophoresis to separate the DNA fragments from several of these highly variable regions. These DNA profiling techniques are also used by scientists in other fields of biology. Conservation biologists use DNA profiling to determine genetic similarity and kinship among populations or individuals. This information is particularly important to captive breeding programs, such as those at zoos, so that the deleterious effects of inbreeding can be minimized. Biologists who study animal behavior use DNA profiling to determine kinship among members of a group and its effects on relationships. The genetic variation revealed by DNA profiling is used by taxonomists to distinguish species. Evolutionary biologists use DNA profiles to compare the similarities and differences among species, constructing hypothetical family trees. Proteins can also be run on gels. Most commonly proteins are run on gels made of polyacrylamide in the presence of SDS. In SDS PAGE (SDS polyacrylamide gel elecrtrophoresis) all proteins are coated with SDS and are thus negatively charged. This technique separates proteins by size. More rarely proteins are separated by charge in gels without SDS. Scientific dyes can also be separated by gel electrophoresis. Like DNA most scientific stains are negatively charged. Scientific dyes are often used in classroom setting to simulate DNA since the chemicals required to visualize DNA in a gel cause cancer. http://www.ehow.com/how-does_5315469_process-gel-electrophoresis.html The Process of Gel Electrophoresis By Donald Miller, eHow Contributor 1. o

Gel electrophoresis makes use of the principle that a substance (genetic material or proteins) will move or migrate through a medium having an electric current. Molecules will move through this medium at differing rates depending upon their molecular sizes and weights. The medium that is used in gel electrophoresis is called agarose gel, which is made from purified seaweed. Slots, called wells, are formed in the agarose gel and the lab workers will place a sample, such as a DNA sample, in these slots or wells. Lab workers also introduce a dye into the sample to make it more visible and easier to trace and record. Once an electric current is applied to the medium (the agarose gel) the molecules in the sample begin to move through the medium. Since the molecules do this at different rates, depending upon their size and weight, observing the streaks or marks left by the dye in the gel allows for analysis. The electric field in the gel medium has a positive pole and a negative pole. In the case of a DNA sample, the DNA contains a phosphate part to its structure and this part of the molecule carries a negative charge. Since opposites attract, this charge results in these molecules being attracted to, and moving toward, the positive pole in the electric field. Again, the smaller molecules move farther and faster in the medium than the larger ones, producing a separation. We can think of it almost as a race. The "winners," the "losers" and those in between spread out into an array that allows the researcher to make identifications. In other manipulations of the process, researchers may place special molecular markers in the medium. These are markers of known weight and characteristics that provide a means of indexing and identifying the sample molecules. Also, the chemical makeup of the medium may be adjusted according to the type of sample being tested. These alterations are done to optimize the conditions to a particular analysis.