Lab 4 - February 12, 2010 Paul Gonzales BIO206L Session:...

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February 12, 2010 Paul Gonzales BIO206L Session: 49540 Lab 4 1) Agarose gel electrophoresis works by separating molecules of DNA by size and charge. An electric field is used to separate positive charged molecules (cations) and negative charged molecules (anions) from one another, and smaller molecules travel further. DNA migrates towards the positive end (anode) because of the negative charge of DNA molecules. The length of DNA can affect the migration through the gel, as well as the conformation of the DNA molecule. Increasing the Agarose concentration also may reduce the migration speed. Increasing the voltage may increase the rate the DNA movies, but voltage is limited because the gel can melt. Xylene Cyanol, Bromophenol Blue dyes, SDS, EDTA, and sucrose are all used as color markers for DNA fragments to track the movement through the gel. 2) Agarose gel once dry will be slightly opaque and light blue in color. It is rectangular in shape and is has a jelly texture to it once solidified. To make an Agarose solution form a gel, you must heat the Agarose solution for about a minute in a microwave and then pour the contents into a tray for thirty minutes until the gel solidifies. Decreasing the concentration of the Agarose will help separate larger sizes of DNA fragments (kb fragments). Lower percentage concentrations of Agarose are better for separating larger DNA fragments because they result in better separation between bands that are close in size. 3) If you wanted your gel to run faster you would need to decrease the ionic strength of the TAE running buffer make it more dilute) and increase the amount of voltage. 4) Xylene Cyanol, Bromophenol Blue dyes, SDS, EDTA, and sucrose are all used as color markers for DNA fragments to track the movement through the gel. With the aid of ultraviolet lighting, DNA appears as orange bands in the gel. 5) Isolated human DNA – 10 µL isolated human DNA, 2.5 µL PRIMER MIX, and 12.5 µL MASTER MIX Plasmid DNA-10 µL plasmid DNA, 2.5 µL PRIMER MIX, and 12.5 µL MASTER MIX Control DNA -10 µL control DNA, 2.5 µL PRIMER MIX, and 12.5 µL MASTER MIX PRIMER MIX = solution of forward and reverse primers MASTER MIX = Taq Polymerase, MgCl 2 , dNTP’s, and 2X buffer The final volume in each tube was 25 µL. The final concentration of the 2X Taq buffer used in a tube with 25 µL of reaction volume is 1X. 6) DNA template – purified DNA used to form single-stranded DNA templates Forward/reverse primers - complementary to target DNA, chosen so not
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February 12, 2010 Paul Gonzales BIO206L Session: 49540 too far apart when they anneal to the DNA, they basically make more DNA by copying DNA from two directions Taq Polymerase – amplifies short segments of DNA, eliminates need for
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Lab 4 - February 12, 2010 Paul Gonzales BIO206L Session:...

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