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Animation: Gel Electrophoresis of DNA

by Pearson
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Gel Electrophoresis of DNA DNA molecules of different lengths can be separated by gel electrophoresis, and the lengths of the DNA molecules can then be determined. In gel electrophoresis, DNA molecules migrate to the positive pole as current passes through the gel, with the shortest DNA molecules moving the farthest. Making the gel is the first step in gel electrophoresis. A common material used for gel electrophoresis of DNA is agarose. Agarose gels are made by first boiling a mixture of powdered agarose and buffer. When the mixture has cooled to about 65 degrees Celsius, the solution is poured into a gel mold. When further cooled to room temperature, the agarose solidifies to produce the gel with indentations called wells. The agarose gel is placed in an electrophoresis apparatus and buffer is added to cover the gel. DNA samples are loaded into the wells using a micropipettor. One sample contains DNA molecules of known lengths so that the experimental DNA samples can be compared to it. Each DNA sample has tracking dyes added to it (usually blue) and either sucrose or glycerol to make the solution dense and sink into the well. After the wells are filled, the power supply is turned on, and the DNA moves towards the positive pole. At this stage, the DNA band or bands cannot be seen, but the tracking dye (dark blue) allows the progress of the electrophoresis to be followed. Smaller DNA fragments move faster down the gel than larger fragments. The DNA is visualized by staining, for example with ethidium bromide, and then destaining in water. The ethidium bromide binds to the DNA and makes it fluoresce orange/pink under UV light. A photograph of the results is taken and used for analysis and for a permanent record of the experiment. The lane on the right has the DNA molecules whose lengths are known; the lengths of these size standards are given as the number of DNA base pairs (bp). The longest DNA molecules move the slowest, so they are at the top; they have 23,130 base pairs. Moving down, the DNA molecules in the bands have 9,416 base pairs, 6,557 base pairs, 4,361 base pairs, 2,322 base pairs, 2,027 base pairs, and, finally, 564 base pairs for the short fast-moving DNA molecules. To determine the sizes of the DNA molecules whose lengths are not known, their positions are compared to the DNA molecules of known lengths. For the sample on the left, the DNA molecules are 6,201 base pairs and 1,268 base pairs. For the sample in the middle, the DNA molecules are 4,796 base pairs, 1,405 base pairs, and 1,268 base pairs. Notice that the shortest DNA fragments in the left and middle samples are the same lengths, indicating that these DNA samples were cut at the same sites (the fragment's ends) with the restriction enzyme that was used.
Gel Electrophoresis of DNA DNA molecules of different lengths can be separated by gel electrophoresis, and the lengths of the DNA molecules can then be determined. In gel electrophoresis, DNA molecules migrate to the positive pole as current passes through the gel, with the shortest DNA molecules moving the farthest. Making the gel is the first step in gel electrophoresis. A common material used for gel electrophoresis of DNA is agarose. Agarose gels are made by first boiling a mixture of powdered agarose and buffer. When the mixture has cooled to about 65 degrees Celsius, the solution is poured into a gel mold. When further cooled to room temperature, the agarose solidifies to produce the gel with indentations called wells. The agarose gel is placed in an electrophoresis apparatus and buffer is added to cover the gel. DNA samples are loaded into the wells using a micropipettor. One sample contains DNA molecules of known lengths so that the experimental DNA samples can be compared to it. Each DNA sample has tracking dyes added to it (usually blue) and either sucrose or glycerol to make the solution dense and sink into the well. After the wells are filled, the power supply is turned on, and the DNA moves towards the positive pole. At this stage, the DNA band or bands cannot be seen, but the tracking dye (dark blue) allows the progress of the electrophoresis to be followed. Smaller DNA fragments move faster down the gel than larger fragments. The DNA is visualized by staining, for example with ethidium bromide, and then destaining in water. The ethidium bromide binds to the DNA and makes it fluoresce orange/pink under UV light. A photograph of the results is taken and used for analysis and for a permanent record of the experiment. The lane on the right has the DNA molecules whose lengths are known; the lengths of these size standards are given as the number of DNA base pairs (bp). The longest DNA molecules move the slowest, so they are at the top; they have 23,130 base pairs. Moving down, the DNA molecules in the bands have 9,416 base pairs, 6,557 base pairs, 4,361 base pairs, 2,322 base pairs, 2,027 base pairs, and, finally, 564 base pairs for the short fast-moving DNA molecules. To determine the sizes of the DNA molecules whose lengths are not known, their positions are compared to the DNA molecules of known lengths. For the sample on the left, the DNA molecules are 6,201 base pairs and 1,268 base pairs. For the sample in the middle, the DNA molecules are 4,796 base pairs, 1,405 base pairs, and 1,268 base pairs. Notice that the shortest DNA fragments in the left and middle samples are the same lengths, indicating that these DNA samples were cut at the same sites (the fragment's ends) with the restriction enzyme that was used.