DNA Profiling Lab
DNA Profiling Lab 18519
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This 2 page Class Notes was uploaded by Shanell Coleman on Monday October 10, 2016. The Class Notes belongs to 18519 at Augusta State University taught by Thomas Buxton in Fall 2016. Since its upload, it has received 7 views. For similar materials see Biology 1101 in Science at Augusta State University.
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Date Created: 10/10/16
Lab #4 DNA Profiling Lab 1. Supernatant: denoting the liquid lying above a solid residue after crystallization, precipitation, centrifugation, or other process 2. Soap: destroying all cell membranes, allowing the cell contents to spill out (cell lysis). 3. DNA is only soluble at a pH near physiological levels. The baking soda serves as a buffering system that raises the pH and releases the DNA from bound proteins. 4. The meat tenderizer: is an enzyme (proteinase) that removes proteins bound to the DNA and destroys enzymes (endonucleases) which would chew up the DNA. 5. Since the supernatant is thick from the cellular contents, carefully pouring the alcohol on top of the supernatant leaves two distinct layers. DNA is soluble in water, but not in alcohol; thus, the DNA present at the water – alcohol interface precipitates out of solution, allowing it to be seen. Gel Electrophoresis Gel electrophoresis is a basic biotechnology technique that separates macromolecules according to their size and charge. It is frequently used to analyze a manipulate samples of DNA, RNA, or proteins. In gel electrophoresis, samples to be separated are applied to a porous gel medium made of a material such as agarose. Agarose is a purified form of agar, a gelatinous substance extracted from red algae. All the samples are loaded into wells of the agarose gel. The gel is then placed in a chamber that is connected to a power supply. To “run” the gel an electrical current is applied to the gel. The chamber is designed with a positive electrode (anode) at one end and a negative electrode (cathode) at the other end. Electrophoresis literally means “to carry with electricity,” once the electric field is established, charged molecules in the samples migrate through the pores of the gel toward their pole of attraction. Molecules with a net negative charge migrate toward the positive electrode and molecules with a net positive charge migrate toward the negative electrode. The overall charge of a molecule affects the speed at which it travels through the gel. Highly charged molecules migrate more quickly through the gel than weakly charged molecules. The mobility of a molecule during gel electrophoresis also depends on its molecular size and shape. The small pores of the gel matrix act as a sieve that provides great resolving power. Small molecules maneuver more easily through the pores than larger molecules and therefore travel relatively quickly. Large molecules encounter more resistance as they make their way through the tiny pores and therefore travel at a slower rate. In order to see the DNA in the gel, a chemical called, ethidium bromide (EtBr) is added to the agarose while it was still in its molten state. Ethidium bromide is a molecule that inserts or intercalates between the bases of double stranded DNA. Under UV light EtBr fluoresces, therefore DNA becomes visible under UV light in the presence of EtBr. EtBr is a potent mutagen (may cause genetic damage), and is moderately toxic. Once the gel has run and a picture of the DNA bands has been taken, you can analyze the size of the DNA bands by comparing it to the lay containing the DNA ladder. A DNA ladder is a solution that contains known sizes of SNA. You compare your samples with the known bands to estimate the size of your DNA bands. A T = Adenine Thymine C G = Cytosine Guanine Bind by hydrogen bonds: each is coiled from double helix nucleotides also are the monomers of nucleic acids. Nucleotides have three components: 1. Phosphate group (same for DNA and RNA) 2. Pentose (5carbons, different sugar for DNA and RNA) 3. Nitrogen – containing base (defines which nucleotide)
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