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Molecular Biotechnology
Bernard R. Glick Molecular Biotechnology Text: Molecular Biotechnology B.R. Glick, J.J. Pasternak and C.L. Patten ASM Press, Fourth Edition
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Biotech Companies Worldwide
in 2004
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Fundamentals of recombinant DNA technology
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Overview of recombinant DNA cloning procedure
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Plasmid pBR322 Strategy for selecting E. coli cells transformed with pBR322
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pBR322 Francisco Bolivar Raymond Rodriguez
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Screening a gene library with a labeled DNA probe (colony hybridization)
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Immunological screening of a gene library (colony immunoassay)
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Screening a genomic library for enzyme activity
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Electroporation Tripartite conjugation
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DNA synthesis and the polymerase chain reaction (PCR)
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The chemical synthesis of DNA
Gobind Khorana
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PCR: first cycle
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Second PCR cycle
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PCR: thirtieth cycle
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PCR amplification of full length cDNA
PCR amplification of full length cDNA. The terminal transferase activity of reverse transcriptase adds mostly dCs to the ends of each full length first strand cDNA
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Directed Mutagenesis and Protein Engineering
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Oligonucleotide directed mutagenesis
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Oligonucleotide-directed mutagenesis with plasmid DNA
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PCR-amplified oligonucleotide-directed mutagenesis
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Error-prone PCR
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Random mutagenesis of a cloned target gene
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DNA Shuffling
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Addition of a disulfide bond between the N and C termini of B
Addition of a disulfide bond between the N and C termini of B. circulans xylanase stabilizes the protein. The activity at room temperature is doubled and it is largely protected against inactivation at high temperature
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Engineering a calcium-independent subtilisn
Engineering a calcium-independent subtilisn. Native enzyme loses its activity when the calcium-binding loop is deleted. After random mutagenesis, several mutants with low activity are isolated. These are combined into one mutant construct with high activity.
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Altering multiple enzyme properties at once
Altering multiple enzyme properties at once. Start with multiple copies of a Bacillus subtilisn gene. Error-prone PCR and then DNA shuffling. Test for high activity at room temp and then stability at high temp
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