Week 07 - Exp 6 Agarose gel

Week 07 - Exp 6 Agarose gel - BIOL 519 Week # 7 - Agarose...

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BIOL 519 1 BACKGROUND: On Tuesday of this week, we will complete the Cloning lab by running samples of the plasmid DNA you isolated and cut with HindIII on an agarose gel. The agarose gel allows us to estimate the size of linear fragments of DNA. Therefore, we can compare the size of the fragments generated in your clones to linear fragments from the original plasmids to determine whether you have a clone of the desired structure. Gel Electrophoresis The phosphate backbone of DNA is negatively charged, and imparts an overall negative charge on the DNA molecule. This negative charge allows DNA to move towards the positive electrode when placed in an electric field. If the DNA is placed into a gel matrix in the electric field, then linear fragments of the DNA can be separated from one another base on their size. Nicked and supercoiled forms of circular DNA can also be separated from one another, although their size can not be determined. Supercoiled DNA is much more compact and therefore migrates more quickly than nicked DNA. The most common gels used to separate DNA are agarose and acrylamide. These gels form a network of linked molecules. The DNA apparently moves through the gel by squeezing through the maze made by the matrix. Small linear molecules can snake through the matrix more quickly than large molecules - and therefore migrate more quickly. Agarose Gel Electrophoresis: Agarose vs. Acrylamide - The choice of agarose or acrylamide for the gel depends mostly on the size of the fragments that need to be separated, and the resolution required. Acrylamide gels are best used for the separation of smaller DNA fragments, in the range of 5-500 bp. In addition, acrylamide gels can resolve DNA fragments that differ from one another in length by only a single bp. The disadvantage of acrylamide gels is that they are somewhat more difficult to prepare, and acrylamide is a neurotoxin and therefore must be handled carefully. Agarose (a highly purified form of Agar - also made from seaweed) gels can be prepared by simply melting the agarose in a buffer solution in a microwave, and then allowing the solution to cool and solidify in a casting tray (basically a mold). Agarose gels do not have as much resolving power as acrylamide gels, but, they have a much greater range of separation - approximately 200 bp -
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BIOL 519 2 50,000 bp. The agarose is used at concentrations ranging from 0.3% (separates the largest fragments) to 2% (separates the smallest fragments). Procedure - The basic procedure for gel electrophoresis is: 1) load DNA into wells at one end of the gel; 2) expose the gel to an electric field - DNA has a negative charge, and therefore migrates toward the positive electrode - with small pieces of DNA migrating faster than large pieces. The dye ethidium bromide is used to visualize the DNA. Ethidium bromide is a fluorescent dye that is planar and can insert itself between the base pairs in DNA. Because it binds tightly to the
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This note was uploaded on 01/22/2011 for the course BIO 519 taught by Professor Ega during the Spring '10 term at Kansas.

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Week 07 - Exp 6 Agarose gel - BIOL 519 Week # 7 - Agarose...

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