04x_Agarose Gel Electrophoresis_040108

04x_Agarose Gel Electrophoresis_040108 - Restriction Enzyme...

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Restriction Enzyme Digestion of gDNA and Visualization Using Agarose Gel Electrophoresis LABORATORY EXERCISE #7x Rev: 3/10/10 Introduction To visualize DNA preparation cut the gDNA with a RESTRICTION ENZYME , e.g. Eco RI. It cuts the DNA only at specific combinations of nucleotides (the sequence GAATTC, figure 1). Among the approximately 3.4 million base pairs in the chromosome of P. aeruginosa that sequence occurs about 500 times. Complete ‘restriction’ with Eco RI will cut the gDNA every time at each of those sites and will always produce the identical series of about 500 fragments. The fragments will be separated using AGAROSE GEL ELECTROPHORESIS and will be visualized by staining with ethidium bromide [EtBr], a dye which inserts itself into the double helix of DNA and fluoresces under ultraviolet irradiation. The pattern of fragments can be photographed (figure 2). The sizes of the 500 Eco RI fragments will range from large (greater than about 30,000 base pairs [bp] {or 30 kilobases [kb]}) to small (smaller than the 600 bp which cannot be resolved in the agarose gel that you will use.) 5' G A A T T C 3' 5' G ........................ A A T T C 3' 3' C T T A A G 5' 3' C T T A A .......... ........... G 5' Figure 1. Restriction of DNA by Eco RI at the palindrome GAATTC. The “sticky ends” created by Eco RI are underlined. Figure 2. Restriction of a sample of gDNA by Eco RI. Lane 1 contains high quality DNA that has been cut into about 500 fragments; lane 2 contains poor quality DNA that did not cut well and ran as a smear.
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