Ex10RestrictionEnzymes(2)

Ex10RestrictionEnzymes(2) - K101 Lab Exercise 10...

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Restriction Enzyme Analysis and Gel Electrophoresis of DNA OBJECTIVES: Learn how to cut DNA into fragments with restriction enzymes. Load and separate DNA fragments by electrophoresis. Determine the size of DNA molecules by use of a Standard Curve. INTRODUCTION In this lab, we will analyze the DNA of small virus called a bacteriophage . Bacteriophages are bacterial viruses that can infect and kill some species of bacteria. They have a simple, linear chromosome that is unique to that particular virus, and like other viruses, can only attack and infect certain bacterial cells. During infection of a bacterial cell, the virus inserts its genome into a bacterial cell in an attempt to ‘hijack’ the bacterial cell’s protein synthesis (transcription and translation) machinery. Bacteria can respond to the threat of bacteriophage infection by producing specific enzymes that chop phage DNA up into small pieces, “restricting” viral infection. Scientists first discovered these enzymes, called “ restriction enzymes ” in the late 1960s and early 1970s. (The bacteria’s own DNA is protected from digestion by methylation of the A and C nucleotides in the DNA). An interesting feature of many restriction enzymes is their ability to recognize a sequence of bases on DNA called a palindrome , defined as a stretch of DNA that ‘reads’ the same forwards (on one strand) as they do ‘backwards’ on the complementary strand. For instance, the figure at right shows the restriction site from one of the first discovered restriction enzymes, EcoRI (“ E. coli Restriction enzyme I”), which recognizes the palindromic sequence GAATTC. Many restriction enzymes cut DNA asymmetrically within the palindrome at a specific site called a " restriction site ", and the asymmetric cut results in small, single stranded regions called “ sticky ends (TTAA in this case). These sticky ends have been used by scientists to join or “splice” together two pieces of DNA that have complementary sticky ends, for instance, to join the human insulin gene cut with EcoR1 to a bacterial plasmid cut with EcoR1, resulting in a recombinant DNA drug called Humulin , made by Eli Lilly. In this lab exercise, DNA from the bacteriophage Lambda (48,502 base pairs in length) will be cut with 3 different restriction enzymes: Pst1, EcoRI, and HindIII, and the resulting fragments will separated by gel electrophoresis . A fourth sample will be the negative control in that is will be incubated without any restriction enzyme. Each of the 3 enzymes recognizes a different palindromic sequence of bases on the DNA, and cuts at specific "restriction sites." [HindIII recognizes the palindrome AAGCTT and PstI recognizes the palindrome CTGCAG]. Palindrome
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Ex10RestrictionEnzymes(2) - K101 Lab Exercise 10...

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