Difference between revisions of "20.109(S07): Agarose gel electrophoresis"
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− | ==Introduction | + | ==Introduction== |
Electrophoresis is a technique that separates large molecules by size using an applied electrical field and a sieving matrix. DNA, RNA and proteins are the molecules most often studied with this technique; agarose and acrylamide gels are the two most common sieves. The molecules to be separated enter the matrix through a well at one end and are pulled through the matrix when a current is applied across it. The larger molecules get entwined in the matrix and retarded; the smaller molecules wind through the matrix more easily and travel further from the well. Molecules of the same size and charge migrate the same distance from the well and collect into a band. | Electrophoresis is a technique that separates large molecules by size using an applied electrical field and a sieving matrix. DNA, RNA and proteins are the molecules most often studied with this technique; agarose and acrylamide gels are the two most common sieves. The molecules to be separated enter the matrix through a well at one end and are pulled through the matrix when a current is applied across it. The larger molecules get entwined in the matrix and retarded; the smaller molecules wind through the matrix more easily and travel further from the well. Molecules of the same size and charge migrate the same distance from the well and collect into a band. | ||
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[[Image:Be109agarosegelsetup.jpg|thumb|left|500px]] | [[Image:Be109agarosegelsetup.jpg|thumb|left|500px]] | ||
− | Today you will separate DNA fragments using an agarose matrix. Agarose is a polymer that comes from seaweed and if | + | Today you will separate DNA fragments using an agarose matrix. Agarose is a polymer that comes from seaweed and if you ve ever made Jell-O , then you already have all the skills for pouring an agarose gel. To prepare these gels, agarose and buffer are microwaved until the agarose is melted. The molten agar is then poured into a horizontal casting tray, and a comb is added. Once the agar has solidified, the comb is removed, leaving wells into which the DNA sample can be loaded. |
− | The distance a DNA fragment travels is inversely proportional to its length. Over time fragments of similar length accumulate into | + | [[Image:Be109gelpercentage.jpg|right|thumb|150px]] |
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+ | The distance a DNA fragment travels is inversely proportional to its length. Over time fragments of similar length accumulate into |