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The Basic Principles of Gene Cloning and Dna Analysis | |
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Why Gene Cloning and DNA Analysis are Important | |
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The early development of genetics | |
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The advent of gene cloning and the polymerase chain reaction | |
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What is gene cloning? | |
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What is PCR? | |
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Why gene cloning and PCR are so important | |
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Gene isolation by cloning | |
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Gene isolation by PCR | |
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How to find your way through this book | |
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Vectors for Gene Cloning: Plasmids and Bacteriophages | |
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Plasmids | |
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Basic features of plasmids | |
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Size and copy number | |
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Conjugation and compatibility | |
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Plasmid classification | |
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Plasmids in organisms other than bacteria | |
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Bacteriophages | |
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Basic features of bacteriophages | |
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Lysogenic phages | |
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Gene organization in l DNA molecule | |
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The linear and circular forms of l DNA M13 - a filamentous phage | |
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The attraction of M13 as a cloning vector | |
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Viruses as cloning vectors for other organisms | |
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Purification of DNA from Living Cells | |
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Preparation of total cell DNA | |
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Growing and harvesting a bacterial culture | |
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Preparation of a cell extract | |
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Purification of DNA from a cell extract | |
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Removing contaminants by organic extraction and enzyme digestion | |
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Using ion-exchange chromatography to purify DNA from a cell extract | |
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Concentration of DNA samples | |
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Measurement of DNA concentration | |
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Other methods for the preparation of total cell DNA | |
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Preparation of plasmid DNA | |
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Separation on the basis of size | |
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Separation on the basis of conformation | |
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Alkaline denaturation | |
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Ethidium bromide-caesium chloride density gradient centrifugation | |
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Plasmid amplification | |
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Preparation of bacteriophage DNA | |
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Growth of cultures to obtain a high l titre | |
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Preparation of non-lysogenic l phages | |
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Collection of phages from an infected culture | |
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Purification of DNA from l phage particles | |
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Purification of M13 DNA causes few problems | |
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Manipulation of Purified DNA | |
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The range of DNA manipulative enzymes | |
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Nucleases | |
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Ligases | |
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Polymerases | |
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DNA modifying enzymes | |
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Topoisomerases | |
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Enzymes for cutting DNA - restriction endonucleases | |
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The discovery and function of restriction endonucleases | |
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Type II restriction endonucleases cut DNA at specific nucleotide sequences | |
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Blunt ends and sticky ends | |
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The frequency of recognition sequences in a DNA molecule | |
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Performing a restriction digest in the laboratory | |
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Analysing the result of restriction endonuclease cleavage | |
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Separation of molecules by gel electrophoresis | |
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Visualizing DNA molecules by staining a gel | |
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Visualizing DNA molecules by autoradiography | |
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Estimation of the sizes of DNA molecules | |
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Mapping the positions of different restriction sites in a DNA molecule | |
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Ligation - joining DNA molecules together | |
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The mode of action of DNA ligase | |
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Sticky ends increase the efficiency of ligation | |
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Putting sticky ends onto a blunt-ended molecule | |
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Linkers | |
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Adaptors | |
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Introduction of DNA into Living Cells | |
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Transformation - the uptake of DNA by bacterial cells | |
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Not all species of bacteria are equally efficient at DNA uptake | |
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Preparation of competent E. coli cells | |
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Selection for transformed cells | |
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Identification of recombinants | |
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Recombinant selection with pBR322 - insertional inactivation of an antibiotic.resistance gene | |
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Insertional inactivation does not always involve antibiotic resistance | |
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Introduction of phage DNA into bacterial cells | |
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Transfection | |
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In vitro packaging of l cloning vectors | |
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Phage infection is visualized as plaques on an agar medium | |
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Identification of recombinant phages | |
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Insertional | |