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Unformatted text preview: Experiment 5 (Lab Periods 5 and 6) Gel Electrophoresis A common method of analysis in molecular biology is Gel Electrophoresis . In general, gel electrophoresis is a process by which the macromolecules within a sample are separated from one another on the basis of size. The process can be applied to different types of macromolecules such as proteins and nucleic acid (DNA and RNA). In this lab you will use gel electrophoresis to determine whether or not the fragment of DNA that you ‘amplified’ by PCR in experiment 4 is the expected size. Although this will not constitute absolute proof, if the fragment present in your reaction tube is the correct size it will strongly suggest that your PCR worked. The basic idea behind gel electrophoresis is that you will force the macromolecules through a porous matrix (the gel). The two most commonly used gels are composed of either agarose - which you will use today - or acrylamide (polyacrylamide gels). DNA is forced to move through the matrix by placing the gel in an electric field. DNA molecules have a negative charge so they will flow from the negative pole to the positive pole of the field. The DNA molecules, however, do not flow freely in the field. Instead, the gel acts like a sieve to impede the migration of the DNA molecules, slowing the rate at which they move towards the positive pole. The key to separating DNA molecules is that larger molecules are more strongly impeded by the gel than smaller molecules. As a result, smaller molecules of DNA (those composed of fewer nucleotides) migrate through the gel at a faster rate. After a period of time, the shorter fragments are further from the origin than are the longer molecules. In this manner, DNA fragments in a solution are separated on the basis of size. There are several basic steps to performing gel electrophoresis that will be described below; 1) Pouring the gel, 2) Preparing your samples, 3) Loading the gel, 4) Running the gel (exposing it to an electric field) and 5) Staining the gel....
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This note was uploaded on 01/30/2012 for the course BIO 2004 taught by Professor Morton during the Spring '11 term at Columbia.
- Spring '11
- molecular biology