full lab 4

full lab 4 - Identification of Vector DNA and Recombinant...

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Identification of Vector DNA and Recombinant DNA utilizing restriction fragment length analysis and transformation of Escherichia coli with selection for ampicillin resistance and b -galactosidase presence. Abstract
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Identification of Vector DNA and Recombinant DNA utilizing restriction fragment length analysis and transformation of Escherichia coli with selection for ampicillin resistance and b -galactosidase presence. Recombinantaly inserted Vector DNA contains substantially different properties than non-recombinant vector DNA. The differences in b -galactosidase functionality and sequence composition allow for identification and differentiation between the two types of DNA. Here we determined the form of plasmid (vector or recombinant) present in two different DNA samples. Restriction fragment analysis was used to determine the presence and location of restriction sites in the plasmid. Bacterial transformation was also used to determine the presence and functionality of gene products in the plasmid. We found DNA 1 to generate fragments of size 4kbp, 2.5 kbp, and 0.8kbp upon exposure to EcoR1 and BamH1, DNA 2 generated fragments of size 2.5 kbp when exposed to any combination of the restriction enzymes. Bacterial transformation of DNA 1 generated 216 white colonies and 9 blue colonies, while DNA 2 generated 400 white colonies and 50 blue colonies. From these results we found DNA 1 to be a recombinantly inserted plasmid vector, and DNA 2 to be unchanged plasmid vector. Future experiments will utilize sequence analysis to determine the exact nature of both the vector plasmid and the recombinant insert. Introduction Characterization of plasmid DNA plays an important role in many biological protocols. Here we attempt to ascertain the nature of two different unknown plasmids by both restriction analysis and transformation analysis in Escherichia coli . The important difference explored here is identification of the plasmid as either vector DNA or recombinant insert DNA. Several differences in properties between
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recombinant plasmid and non-recombinant plasmid exist for exploitation in analysis, such as the presence and functionality of ampicillin resistance, b- galactosidase presence and functionality and plasmid size (Kosinski-Collins, 2007). Restriction analysis of plasmid size relies on the principle and usage of restriction enzymes. Restriction enzymes are proteins normally produced by cells as an agent against invasion by a foreign virus. Restriction enzymes are a nuclease enzyme; they cut the phosphodiester bond in double stranded DNA. Most restriction enzymes are highly specific for certain DNA sequences and these sequences are palindromic (read the same in both the 3’ to 5’ and 5’ to 3’ directions). Since restriction enzymes cut at specific sequences, DNA treated with a nuclease generates a specific number and size of smaller DNA fragments that are semi-unique to a DNA or plasmid sequence (Sharp, 1973). The simplest and least specific method to determine the number and size of DNA fragments is to
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full lab 4 - Identification of Vector DNA and Recombinant...

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