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DNA Gel Electrophoresis

Nathan Walsh Biophysics 03/15/2012

Introduction of Gel Electrophoresis


The information that determines physical traits in found in our DNA. The base pairs in DNA code the information needed to do everything from created proteins, to determining the color of our hair. With the information in the DNA we could have the possibility to prevent genetic diseases, be preemptive in the treatment of the ill, or genetically engineer the perfect human. This task is daunting do to the fact that the human genome is made up of approximately three billion base pairs. Because these pairs are two small to observe under a microscope, other methods are used to determine the sequence of the DNA. One of the earliest method was gel electrophoresis. Initially a very slow process, the method has been advanced to a more efficient method known as cyclic array sequencing that can sequence many DNA in a single run. Because the genome is so large, a restriction enzyme is used to cleave the DNA between base pairs. By knowing what type of enzyme is being used the experimenter knows where the DNA is cleaved because the Enzyme cleaves at a specific nucleotide sequence. The creates smaller strands of DNA that are ready for sequencing. Though this method is rather old, it is still used by biologists, chemists and physicist because of the ease of sample preparation. The downside to gel electrophoresis is that the DNA must be cloned into millions of samples, then cut, so the original DNA is lost. More advanced methods are currently being perfected that will potentially sequence the DNA much faster, but this has yet to be achieve at the accuracy of gel electrophoresis.

Principle of the Measurements


DNA has a slight negative charge because of its phosphate backbone. Because of this slight charge, the DNA will migrate in an electric field toward the positive anode. This is known as electrophoresis. This migration is rather fast and must be slowed for observing. The gel in gel electrophoresis is an agarose gel derived from sea weed. When set, the gel forms a matrix of small pores that the DNA can migrate though, but because of this matrix the DNA is slowed. The speed of the DNA is dependent on its charge and the viscous drag due to the gel. We can determine the acceleration of the DNA by balancing the magnetic and drag forces using Newton's Second Law. (1) Here the migration force is given by qE. The drag force is dependent on the velocity of the particle, the shape of the DNA, and the viscosity of the gel. These parameters are difficult to determine. When the forces are balanced there is no acceleration and the DNA is at a constant maximum velocity. This maximum velocity is reached rather quickly. Because of size, the

smaller DNA lengths or shapes experiences less drag force and thus attains a higher max velocity. A potential is applied and after a given amount of time the gel is removed from the electric field and a dye is added so that the DNA will fluores. If we know the distance traveled and the time this took, then an average velocity can be determined and from this velocity, the characteristics of the DNA can be determined. Because the viscous drag constants are difficult to measure, it is more common to compare the un-sequenced DNA sample to a sample of known length. When the DNA is loaded into the wells of the gel, a ladder marker is loaded into the gel in one well. This ladder marker has many different length of DNA of known size and allows the experimenter to compare the distance traveled by their DNA to the distance travel by DNA of known length, thus determining the length of the DNA sample. Knowing the lengths of the sample and the location of enzyme cleaving, we determine the sequence of the base pairs.

Materials and Equipment needed


Microliter pipetter Small eppendorf tubes for preparing samples. Dried agarose Concentrated TBE and TE solutions Ethidium Bromide Gel mold Electrophoresis box DC power supply UV light box with safety glasses Camera

Procedure to Perform the Experiment


1. Prepare the agarose gel. 1.1. Take stock buffer of 10x TBE buffer and dilute with water to obtain 50mL of 1x buffer for agarose. 1.2. Make an agarose solution that is 1% by weight of agarose using the 50mL of 1x buffer 1.3. The agarose will dissolve in heat. Microwave the agarose solution until all solid is dissolved. 1.4. Add L of Ethidium Bromide to the agarose solution. This will alter bind with the DNA and cause it to glow under UV light, known as fluorescence. 1.5. Pour the gel into the mold. Place the well comb into mold and allow to solidify for approximately 30 minutes. 1.6. While agarose hardens, prepare DNA samples. 2. Prepare DNA samples. 2.1. Approximately 50ng of DNA is needed for each sample. With a total final solution volume of 12L. 2.2. Prepare 10L of DNA solution using the appropriate amount of DNA to achieve 50ng and then TE buffer to volume.

3. 4. 5. 6. 7. 8.

2.3. Next add 2L of 6x loading dye to each sample to aid in placement in wells. Fill the Electrophoresis box with 1x TBE buffer and place the loaded gel in the box so that it is slightly submerged. Connect electrodes and run the gel at 90V and 80mA for the predetermined time. Remove gel from electrophoresis box and place on UV lightbox. Using UV goggles, examine the gel and take a picture to use later. Clean up all solutions and samples, and place the chemicals back where they were found. Analyze the photo using the ImageJ software to determine total distance covered by DNA leading to determining the characteristics of the DNA.

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