As seen on many crime shows and in real-life crime scenes, it is necessary to be able to identify DNA. Most of the time, this is done using a technique known as gel electrophoresis. Gel electrophoresis is a method used to separate the macromolecules that make up nucleic acids, such as DNA and RNA, along with proteins. Gel electrophoresis is significant because it has given scientists insight on what cells cause certain diseases and has led to advancements in DNA and fingerprint identification. My experiment will use gel electrophoresis to compare the separation of food dye in different agarose concentrations. The background for this experiment includes the following subjects: inventors, real-world uses, necessary components, separation, …show more content…
This theory was formulated in 1970 and is one of the most recent on the topic of gel electrophoresis. The National Academy of Science states, "The behavior of macromolecules in gel filtration and gel electrophoresis may be predicted from Ogston's model for random mesh work of fibers. This model has been generalized to apply to non-spherical molecules and to several gel types. The theory defines conditions for optimal separation and optimal resolution in gel electrophoresis and gel filtration" (Unified Theory for Gel Electrophoresis and Gel Filtration). There are a few components that compose a gel electrophoresis chamber. First of all, there is a chamber used to contain the gel. The gel used in my experiment will be made from agarose. In order to create an electric field, there are positive and negative electrodes attached to a power source. My experiment will use alligator clips as the electrodes and nine-volt batteries as the power source. A buffer solution, in this case a 1% solution of baking soda, submerges the gel to allow the ions to carry through and maintain the stability of the Ph levels. A comb is used to generate wells inside of the gel to hold the samples. The comb will be made of styrofoam (Forensic Science: Building Your Own Tool For Identifying DNA).
Proteins, or nucleic acids, are isolated and separated within a gel. The gel is cast in a thin, welled slab. In this particular experiment, the gel will be
A number of steps are required to isolate DNA from cellular contents. Describe what happens at each step, and why it acts to separate the parts of the cell. /6
Reaction Tube Reaction Buffer DNA 1( μl ) DNA 2 ( μl ) Enzyme 1 ( μl ) Enzyme 2( μl ) Final Volume ( μl ) Crime Scene Samples Crime Scene DNA, cut with enzyme 1 ready for electrophoresis X -- 45* Crime Scene DNA, cut with enzyme 2 ready for electrophoresis -- X
Figure 1 contains gel electrophoresis for protein samples. The lanes were labeled from 1 to 10 from the right to the left. Lane 1 contained the ladder fragment. Lane 2 contained the filtrate. Lane 3 contained the S1 sample. Lane 4 contained the P1 sample. Lane 5 contained the P1 medium salt sample. Lane 6 contained the P1 high salt sample. Lane 7 contained the S2 sample. Lane 8 contained the P2 sample. Lane 9 contained the P2 medium salt sample. Lane 10 contained the P2 high salt sample.
The idea behind Gel Electrophoresis is that we inject a slab of gel with the DNA we found at the crime scene. We then inject the same gel, next to the crime scene DNA, with suspect 1’s DNA and suspect 2’s DNA. We then send an electric current through the gel and wait for the results. The smaller molecules in the DNA will travel farther than the bigger molecules because the bigger ones will have difficulty making its way through the microscopic beads in the gel. After the separate bands appear in the gel, we stain it with a special chemical called Ethidium Bromide to give it a color under the blue
The proteins are also added to a Laemmli sample buffer in order to give each protein a negative charge so it is able to get pulled through the polyacrylamide gel. The next step is to put the gel into the electrophoresis module and to run it. It is run until the proteins have almost reached the bottom of the gel. A blue tracking dye is added to the Laemmli sample buffer in order to track the distance in which the proteins travel through the gel. If it is run for too long, the proteins will run off the bottom of the gel and it will mess up your results. Once the protein reach the bottom of the gel, the gel is stained in order to be able to see the individual bands of the different proteins. When the gel is stained, the protein distances will be able to be measured and compared. For a detailed procedure, refer to the Comparative Proteomics Kit I: Protein Profiler Module Lab Manual.
Using the electric current, scientists pass the DNA through gel, and as smaller molecules get through gel quicker than those of bigger sizes, DNA molecules get separated according the sizes of the molecules. We utilize the property that large molecules move slower, and DNA is slightly negatively charged(due to phosphate groups), so it will move to the positive pole of the gel.
After electrophoresis was finished, my boss removed the gel casting tray from the running chamber. Then, she carefully transferred the gel to a DNA staining tray. My lab assistants and I were going to stain it so it would be easier to see the
Once the protein goes through the pores of the gel based on negative charge, if the charge is high, then the faster it moves in an electric field. The shape is important to know based on shape how the protein would travels through it, for example a round shape will make the protein move faster than rod shape due to the rod will bend which this will cause the protein to stop. The size of the is important to determine whether its big or small if its big then the protein are able to have difficulties to get through the pores and move slowly. The protein travel differently than other natural protein in the gel. The mutation change impacts the negatively charge amino acid with hydrophobic amino acid, which cause the protein to change the charge and shape. This process is to either introduce or eliminate a restriction enzyme, which allows it to eliminate the pieces of DNA whether they have it or not. Each of the restriction enzymes is able to identify and eliminate it, which cause the DNA not a normal DNA because of the size of fragments that can be apart from using gel electrophoresis. This causes the DNA fingerprint to change by different pattern on the
3. For this experiment, your sample was applied to a cellulose acetate strip and the migration in an electric field was measured at various pH values. There are other methods that can be used during an electrophoresis experiment. Discuss one alternative electrophoresis method and compare and contrast it to the method used in lab this week.
Purpose The main purpose of this experiment was to test and observe how DNA molecules are being tested or separated. Introduction The final goal of this lab was to successfully measure the size of different samples of DNA placing each samples into a well in agarose gel and running a current through a charged chamber.
After the macromolecular material has been introduced to the selected matrix within a controlled apparatus, essentially a shallow plastic box, electrical nodes attached to opposite ends of the apparatus supply the matrix with a positive and negative electrical current, respectively. Because of phosphate groups present in nucleic acids, DNA and RNA possess a natural negative charge and will be opposed by the applied negative current. The macromolecules will then begin to migrate away from the negative node and toward the positive node along a horizontal pathway. As the macromolecules migrate, their varying components will be caught and separated within the pores of the charged matrix. In this sense, the matrix acts as a sieve rendering the components discrete for analysis. Proteins, moreover, behave identically to nucleic acids during electrophoresis in terms of reactivity, but require treatment with sodium dodecyl sulfate prior to electrophoresis. This detergent binds with polypeptide chains within the protein and imparts it an overall negative charge. The protein is then opposed by the negative charge and migrates toward the positive charge within the apparatus. Multiple samples may be tested simultaneously within the electrophoresis apparatus because each box contains small buffer lanes or "wells" that separate the samples within the matrix. This allows for
The percent of agarose gel used in the lab is .8%. The amount of agarose used makes a difference in the fingerprinting procedure because since agarose gel is the environment around the polymers, (aka the matrix) then the higher the percentage of agarose, the denser the matrix. Also, the denser the matrix, the harder it is for the DNA to pass it when it travels to the positive
Each lab bench will make, and run one gel electrophoresis per table. Once the gel is ready to be loaded, load five microliters of PCR DNA ladder into the first well, as a standard. This should be found in a tube in and ice bucket. Next add two microliters of 6x loading dye into the six sample tubes. The dye should be mixed in thoroughly by gently pipetting up and down after adding the dye. Following that you should load fifteen microliters of each sample into the following six wells. Since lane one will have the DNA ladder lane two starts the samples using the orange tube, then the blue, yellow, red, green, and pink tubes go into lanes three, four, five, six, and seven respectively. Once all the samples are loaded turn on the electrophoresis machine, and wait until the bromophenol blue tracking dye has migrated at least half the length of the gel. Lastly using gloves carefully remove the gel and carry it to the UV light box to view, and photograph the gel (Hass C., Woodward D., and Ward A., 2010.).
Isolation of DNA is mainly done by adding different other digestive enzyme which reacts with constituents in the sample encountered at the crime scene, other than DNA. The different enzymes are proteases which breakdowns amino acids and proteins RNAase which digest RNA particles thus leaving only DNA in the test-tube. Ice cold ethanol is added to this homogenate to precipitate DNA. The DNA can be isolated slowly stirring a glass rod which is known as spooling.
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of DNA or proteins in a matrix ofagarose. The proteins may be separated by charge and/or size