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Unformatted text preview: BIOL 519 Week # 4- Cloning - Restriction Digestion 1 BACKGROUND: This week, we will begin a cloning experiment that will continue for 3 1/2 weeks. Over the course of this experiment, you will learn a variety of basic techniques that are central to molecular biology and the cloning of DNA. A clone can be defined as a collection of DNA molecules or organisms that are genetically identical to one another because they result from the asexual replication of a single original DNA or organism. We will not be cloning sheep. Instead, we will clone a fragment of DNA - specifically, a gene encoding resistance to the antibiotic . When cloning a fragment of DNA, the goal is to insert the DNA fragment into some other DNA (the vector) that is capable of being replicated (has an origin of replication). When cloning into bacterial systems, the vector is generally either a plasmid or a phage. The DNA cloned may be of any origin - bacterial, human, plant, or sheep, for example. Once the DNA fragment is inserted into the vector, and the resulting clone is transformed (or moved by some other suitable genetic exchange mechanism) into an appropriate host (a bacterial strain in which the plasmid or phage can replicate), the cloned DNA fragment will be copied each time the bacterial cell divides, and a nearly infinite supply of the cloned DNA fragment is available. In our experiment you will clone a gene encoding resistance to the antibiotic into a plasmid vector. We will then transform the DNA (hopefully containing some clones) into an E. coli strain that is specially designed to transform well, and allow the isolation of high quality DNA upon re-isolation. We will use the phenotype of the colonies to identify those that are likely to contain the desired plasmid clone. We will then re-isolate the DNA from the suspected clone-containing colonies, and analyze the DNA to determine whether it has the structure we would predict. I will introduce detailed background for each of the steps in the cloning procedure as needed. This week, you will be performing Restriction Digestions and Ligations. Restriction and Modification Systems- Restriction digestion of DNA utilizes naturally occurring enzymes called restriction endonucleases. These enzymes are naturally encoded in bacterial cells, presumably to protect the bacteria from DNA that might try to invade the bacterial cell, such as phage. The most commonly used restriction endonucleases recognize a particular sequence in DNA (usually 4, 6, or 8 bp long), bind to that sequence, and then cleave the DNA within the sequence. The sequence that is recognized is most often a palindrome (reads the same backwards and fowards). An example of a palindromic recognition sequence is the sequence recognized by the enzyme Hin dIII: Hin dIII: 5-AAGCTT-3 3-TTCGAA-5 While restriction endonucleases are useful to destroy invading DNA, they would also destroy the bacterias own DNA without some form of self protection. bacterias own DNA without some form of self protection....
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- Spring '10