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| Preface | |
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| Course Synopsis: Introduction | |
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| Safety Precautions | |
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| Daily Schedule | |
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| Acknowledgements | |
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| Exercises: I DNA Preparation, Polymerase Chain Reaction, and Molecular Cloning: Cesium chloride-ethidium bromide density gradient centrifugation | |
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| PCR to synthesize virD2 flanked with restriction sites | |
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| Restriction digests of plasmid pGEX2 and PCR products | |
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| Purification of DNA fragments from agarose | |
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| Ligation of PCR product to pGEX2 vector | |
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| Transformation of E. coli with the ligated plasmid | |
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| Small-scale preparation of plasmid DNA by the alkaline lysis method | |
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| Restriction analysis | |
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| Study questions | |
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| Protein Expression, Purification, and Analysis: Expression and purification of a fusion protein | |
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| SDS-polyacrylamide gel electrophoresis | |
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| Silver stain detection of proteins | |
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| Study questions | |
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| Oligonucleotide-Directed Mutagenesis: Restriction digests of virD2 (in pCS64) and pUC119 | |
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| Purification of DNA fragments from agarose | |
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| Ligatation of restriction fragment and vector | |
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| Transformation of E. coli with the ligated plasmid and recovery clones | |
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| Small-scale preparation of plasmid DNA from broth cultures | |
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| Restriction digest of DNA: examination to confirm insert | |
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| Preparation of single-stranded DNA template | |
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| Phosphorylation of oligonucleotide | |
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| Annealing mutant oligonuleotide to template | |
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| In vitro DNA synthesis by primer extension | |
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| Transform synthesis reaction into E. coli DH5a | |
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| Small-scale preparation of plasmid DNA | |
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| Confirmation of mutants by restriction analysis | |
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| Study questions | |
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| DNA Sequencing: Polyacrylamide sequencing gel electrophoresis | |
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| Didexy sequencing | |
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| Automated Sequencing | |
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| Introduction to databases and gene sequence analysis | |
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| Study questions | |
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| Southern Blot Detection of DNA: Preparation of genomic DNA from Agrobacterium tumefaciens | |
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| Restriction digestion of genomic DNA | |
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| Agarose gel electrophoresis of restriction fragments | |
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| Southern blot: denaturation and blotting of DNA gel | |
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| Preparation of probe nick translation | |
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| Hybridization and washing of Southern blots | |
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| Study questions | |
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| Northern Blot Detection of mRNA: Preparation of RNA from tobacco leaves | |
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| Agarose-formaldehyde gel electrophoresis | |
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| Northern blot: denaturation and blotting of RNA | |
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| Probe preparation | |
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| Hybridization and washing of Northern blots | |
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| Study questions | |
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| Protein Interaction Analysis in Yeast: Yeast transformation | |
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| Filter b-galactosidase assay | |
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| Index | |