lab report 1-Sahil-LT - Single and Double Restriction...

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Single and Double Restriction Enzyme Analysis Using E. coli Plasmid DNA By Sahil Amin [email protected] Biology 230W 17 November 2014 Abstract In 1953, the double helix model of DNA was proposed by James Watson and Francis Crick. This served as a watershed moment in biology, as the ability of DNA to store and replicate genetic information via daughter generations gained clarity. In modern times, biologists are aware the genetic information can be stored in chromosomal DNA as well as plasmid DNA, found uniquely in bacteria. Plasmid DNA extraction and analysis is a vital step in techniques such as gene cloning. In this lab, Escherichia coli were used for plasmid DNA extraction, and were cut using restriction enzymes. While transformation using pGLO did not occur, due to unforeseen
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circumstances, a hypothetical pGLO transformation will be used to discuss the results of a gel electrophoresis. Introduction Plasmids are extra-chromosomal molecules found in bacteria. Plasmids hold genetic information and are often circular. Because a plasmid lies outside the chromosome, it requires its own unique origin of replication. To maintain stock, plasmids are often selectively chosen based on selectable markers. Selectable markers are arbitrary definitions given to plasmids that either confer a selective advantage or disadvantage to given bacteria. Cloning sites within plasmids provide easy access points for restriction enzymes to cleave and allow uptake of new genetic information. Transformation, or uptake of foreign DNA by bacteria, occurs by introduction of ligated plasmids into bacteria colonies. Newly transformed colonies should already be antibiotic resistant and then screened for successful transformation. Using gel electrophoresis, the size of restriction fragments released from the plasmid can be checked to verify transformation. In this lab, transformation of E. coli was not performed; however, stock bacteria containing pGLO will be used later in the discussion. pGLO is genetically engineered to contain numerous reporter genes, as well as the green fluorescent protein, GFP. GFP originally was isolated from the jellyfish Aequorea victoria. Under ultraviolet light, GFP glows green.
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Figure 1. Above is a sample plasmid as illustrated by an author. The plasmid contains markers for restriction enzymes and a selectable marker gene.
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