Gel Electrophoresis: Separating DNA and RNA

1. If each individual has such a small amount of DNA, how do the bands on the gel contain enough DNA to be visible?

charged DNA to bind the positively charged minicolumn. 2 ml of column wash is then used to

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.

DNA samples can be extracted from hair, blood and skin. Siblings have different DNA fingerprints because everyone has 23 pairs of chromosomes and for each pair one is one of your mother’s chromosomes and the other is your father’s chromosomes. Other than identical twins no other people have exactly the same DNA. DNA fingerprinting is used every day to determine whose parents or siblings are whose, it is used in crime scenes to determine who was at the scene, can be used to determine where a certain inherited gene is inherited from and it can be used to identify a body that is deceased. During electrophoresis an electric current passes through the agarose gel, therefore moving the DNA samples through the gel. The smaller the DNA fragment the faster it moves through the gel. The finished product will look like a series of bands, some will match up and some won’t. The different bands in electrophoresis represent different gene fragments.

Agarose is a polysaccharide that is used especially in gels for a process called electrophoresis. It is used in DNA fingerprinting because it slows down the speed at which the DNA migrates from the negative pole to the positive pole so that the process is easier to visualize and to gather results from. In addition to this, the agarose also creates the actual gel that is being manipulated.

Being able to gather large amounts of DNA together has many potential advantages. It is possible to collect this DNA and use it for purposes such as research for medical uses. To extract the DNA from cells, you need to follow a series of steps which will result in the separation of DNA from other parts of the cell. After following the procedure, you will be left with a stringlike mass of DNA that can be easily examined.

There are many different ways that one can extract DNA from biological evidence. Forms of biological evidence are hair cells, tissues, blood, semen, skin cells, muscles, brain cells, bone, hair, waste, and finger nails. Each involves similar but different extraction techniques. Extraction is a technique in which out of the entire sample, DNA is the only thing left to have ("DNA Extraction”). There are four different steps when it comes to extracting the DNA, preparing the sample, binding the DNA to the membrane, wash, and elution of pure DNA (Kennedy). Preparing the sample involves, taking the sample and putting into a microcentrifuge tube and adding buffers into the sample. Binding the DNA to the membrane is important because when the DNA sample is transferred over to the column there is silica (Kennedy). In the binding stage, ethanol is added which allows for the nucleic acids to stick to the silica more, allowing the DNA to bind. The next stage is the wash stage, the wash stage uses a lot of buffers

Electrophoresis was performed using an electrophoresis system (WEALTEC Corp., USA.) at ambient temperature. The current flow maintained at 20 mA until the dye front reaches to the bottom. VII. The gel washed with ultra pure water, placed in fixing reagent on a shaker for 30 min, and again washed the gel with ultra pure water.

The function of the gel is so that the DNA strands can separate out. The electrophoresis process is necessary because, due to DNA having a negative charge naturally it will be attracted to substances that are positively charged, so by positioning the gel, with the DNA samples injected into its wells, facing towards the positive end of the electrophoresis apparatus with the DNA samples on the opposite end closest to the negative end, the DNA strands with gradually move down the gel and separate based on their band size. Before separating the DNA strands can be accomplished, the gel must first be made accordingly as well as the samples. In order to make the gel, agarose powder and a buffer must be mixed and cooked together first. The buffer that was used in this experiment is the 1X TAE buffer. Utilizing a buffer is important in making the gel for gel electrophoresis because “gel electrophoresis depends upon the effective maintenance of pH within the matrix” (Magdeldin). Moreover, while providing the correct pH conditions, it also has the correct salt conditions for DNA polymerase. Salt is important

The purpose of the gel electrophoresis lab was to separate DNA and RNA on a gel matrix to figure out the process of DNA fingerprinting. For this lab the materials used to solve the problem that was given I used a prepared gel, sample loading device, evidence samples, and a loading buffer.

DNA isolation is a process of DNA purification through physical and chemical means. DNA was protected within the nuclear membrane of B. rapa, which was surrounded by a cell membrane and cell wall. Mechanical and chemical lysis of the cell was necessary to break open the cells and solubilize the membranes to isolate the DNA. Mechanical lysis, which consisted of grinding the leaves with a pestle, broke down the cell wall. Chemical lysis, through the use of lysis solution, broke down the lipid-base membranes. The lysate was placed inside the Zymo-spin IV spin filter to remove chunks of debris from the solubilized cell components. A binding buffer was added to the collection tube that held DNA,

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

To sequence DNA it needs to be amplified first and this can be done in two ways; The Polymerase Chain reaction (PCR) and Cloning. Cloning isolates the DNA fragments from restriction enzymes and then combines the fragments with the vector. Once united with the vector the DNA fragments are then able to replicate to provide an unlimited amount of DNA for the experiment. PCR allows DNA to be multiplied millions of times in a couple of hours, PCR is used because it’s highly specific, easily automated and can amplify very small samples of DNA. The technique of PCR has had a huge effect on genetic disease diagnosis. To sequence the DNA, Gel Electrophoresis is used as it separates DNA fragments according to their size by running an electric current through the gel (Boehm). Mapping and sequencing the genome of organisms has allowed tests to be conducted, for example; gene tests, this is where individual genes or short lengths of DNA or RNA are tested for and Chromosome tests; this is where whole chromosomes or long lengths of DNA are tested for (National Human Genome Research Institute, National Institutes of Health 2014c).

In Figure 1, the photograph shows how each of our samples ran in the agarose gel. We can see different bands in different wells, meaning that the enzymes were working and that the plasmid has been cut in different patters according to the reaction. Before starting measuring the distances and doing the calculations, it is noticeable that the bands of the ladder were not very well separated. Therefore, it will not be accurate to rely on those bands to make the standard curve and determine the sample fragment sizes. The unclear ladder band could be a result of different factors, including the amount of time the gel was running for, the components of the buffer, the gel concentration or even the voltage and current conducted. Therefore, we can say that the digestion had worked, since we are able to see different fragments in the samples, but the electrophoresis was not very successful as the ladder bands were not separated very well, in addition to their general blurriness.