BIOL 1406

PreLab 9b.1

After cutting DNA with a restriction enzyme, how can I use agarose gel electrophoresis to analyze the DNA fragments?

During exercise 9a, you were given 3 unknown plasmid samples labeled A, B, and C. These samples were used to carry out 2 experiments:

1. Competent E. coli cells were mixed with the 3 unknown samples in order to allow transformation to take place. The E. coli cells were then plated on nutrient agar containing ampicillin and Xgal.
    
2. The restriction enzyme EcoRI was mixed with the 3 unknown samples in order to cut any DNA present into fragments. The resulting DNA fragments were then separated using agarose gel electrophoresis.

In this lab, you will view the results of these 2 experiments in order to determine which unknown sample had no plasmids, which sample had non-recombinant pUC18
plasmids, and which sample had recombinant pUC18 plasmids. In addition, you will use the results of agarose gel electrophoresis to estimate the size of the fragments that were produced when EcoRI digested the DNA in the 3 unknown samples.

The separation of DNA molecules by agarose gel electrophoresis is similar to the separation of proteins by SDS-PAGE electrophoresis. In both cases, the molecules to be separated are loaded onto a gel and then subjected to an electrical field. The cathode (negative electrode) attracts positively charged molecules, while the anode (positive electrode) attracts negatively charged molecules. This attraction causes the molecules in the mixture to migrate through the gel at different rates depending on their size, shape, chemical composition, and electrical charge. As the molecules migrate at different rates, they gradually separate from each other. The main difference between protein electrophoresis and DNA electrophoresis lies in the support medium. While polyacrylamide gels are generally used for protein separations, agarose gels are commonly used for DNA separations. Agarose is a polysaccharide that is extracted from seaweed. Agarose gels are cast by dissolving agarose in a Tris-buffered solvent at a high temperature, and then pouring the solution into a horizontal tray and allowing it to cool. A comb is inserted into the cooling gel so that wells are formed as the gel solidifies. The cooled gel is then transferred into a gel box where it is submerged in a Tris-buffered electrode solution before loading DNA samples into the wells. Since these gels are run horizontally and beneath the electrode buffer, they are sometimes referred to as “horizontal gels” or “submarine gels”.

Lab set-up for agarose gel electrophoresis

DNA molecules will migrate towards the anode during electrophoresis because they have a high concentration of negatively-charged phosphate groups in the sugar-phosphate backbone of the double helix. These negative charges provide a fairly uniform charge-to-mass ratio among all DNA molecules. Because of this uniform ratio, the migration rate of DNA molecules during agarose gel electrophoresis is almost entirely a function of size. As with protein electrophoresis, smaller molecules move through the gel more rapidly than larger molecules. Scientists generally measure the size of DNA molecules in terms of number of base pairs (bp) rather than molecular weight (MW).

DNA, like most proteins, is colorless. Therefore, before DNA samples are loaded onto an agarose gel, they are mixed with a sample buffer that includes a blue tracking dye. During electrophoresis, the tracking dye migrates very rapidly through the gel, along with the smallest of DNA fragments. When the dye approaches the end of the gel, you know it is time to stop the electrophoresis. Glycerol is also included in the sample buffer in order to make the sample denser so that it settles into the well as it is loaded.

After electrophoresis is complete, the gel is stained so that the DNA bands can be seen. The most commonly used dye for staining DNA is ethidium bromide. DNA bound to ethidium bromide fluoresces strongly under ultraviolet light, so the gels are viewed and photographed using ultraviolet light. However, because both ethidium bromide and ultraviolet light are mutagens, you will be using a less sensitive but safer stain for your DNA gels: methylene blue. Methylene blue is a blue-colored stain that binds to DNA in the same way that Coomassie Blue binds to proteins. The blue color is easily seen using normal visible light Recall that during SDS-PAGE electrophoresis there is a linear relationship between the migration distance of the proteins and log of MW. Similarly, during agarose gel electrophoresis there is a linear relationship between the migration distance of the DNA molecules and log of bp (number of base pairs.) Because of this, agarose gel electrophoresis can be used to estimate the size (number of base pairs) of DNA molecules. In order to do this, a standard curve that shows the relationship between migration distance through the gel and log of bp must be generated. The standard curve is generated by measuring the migration distance of several DNA molecules of known size (number of base pairs), plotting a scatter diagram of migration distance vs. log of bp, and then using linear regression to determine the equation of the “best fit” straight line for the data points. Once this is done, you can substitute the migration distance of any DNA molecule on your gel into the linear regression equation and then calculate the log of bp for that molecule.

Agarose gel showing DNA bands stained with methylene blue. Sample "A" was loaded in lane 2, "DNA markers" were loaded in lane 3, sample "B" was loaded in lane 4, and sample "C" was loaded in lane 5.

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