Introduction to the Biotechnology Laboratory.- 1.1Overview.- 1.2 Background.- Scope of Biotechnology.- Working in the Biotech-nology Laboratory.- Laboratory Safety.- Notes, Records, and Labels.- Housekeeping.- Preparation and Storage of Buffers.- Preparation and Storage of Media.- Care and Maintenance of Cultures.- General Rules for Protein Handling.- Storage of Nucleic Acids.- 1.3 Experimental Design and Procedures.- Preparation ofYPD and YPG Media.- Streak Plating of Escherichia coli and Saccharomyces carlsbergensis.- Microscopic Observation of Bacteria and Yeast.- Inoculation of YPD and YPG with Saccharomyces carlsbergensis.- Study Questions.- Further Readings.- 2. Introduction to Proteins.- 2.1 Overview.- 2.2 Background.- Protein Function.- Characterization of Proteins.- Measurement of Biological Activity.- 2.3 Experimental Design and Procedures.- Time Course Study of ?-Galactosidase Assay.- Analysis of Culture Broth for a-Galactosidase Activity by the Standard a-Gal Enzyme Assay.- Colorimetric Protein Assays.- Biuret Protein Assay.- Lowry Protein Assay.- Bradford Protein Assay.- Preparation and Inoculation of YP Broth for ?-Galactosidase Production.- Study Questions.- Further Readings.- 3. Protein Isolation and Preparation of Crude Extract.- 3.1 Overview.- 3.2 Background.- Selection of Protein Source.- Cell Disruption Techniques.- Factors Affecting Activity in the Crude Extract.- 3.3 Experimental Design and Procedures.- ?-Gal Assay of Yeast Culture Broth.- ?-Gal Assay of the Yeast Supernatant.- Preparation of Yeast Protoplasts.- Lysis of the Yeast Protoplast Cells.- Collection of Supernatant and Determination of Total Protein.- Stability Study of Yeast Supernatant.- Study Questions.- Further Readings.- 4. Batch Purification of Proteins.- 4.1 Overview.- 4.2 Background.- Bulk Precipitation Techniques.- Batch Protein Capture.- Gel Binding Capacity.- 4.3 Experimental Design and Procedures.- Determination of Binding pH.- Capture of ?-Galactosidase by Batch Ion Exchange.- Study Questions.- Further Readings.- 5. Protein Purification by Column Chromatography.- 5.1 Overview.- 5.2 Background.- Components of a Chromatography System.- Chromatographic Resolution.- Gel Filtration Chromatography.- Ion Exchange Chromatography.- Hydrophobic Interaction Chromatography.- Affinity Chromatography.- Chromatofocusing.- 5.3 Experimental Design and Procedures.- Desalting of Batch Purified ?-Galactosidase.- Preparation of the Ion Exchange Column.- Chromatography of the Desalted Protein Sample.- Analysis of lEX Purified ?-Galactosidase with the Lowry Protein Assay.- Study Questions.- Further Readings.- 6. Protein Analysis and Verification.- 6.1 Overview.- 6.2 Background.- Gel Electrophoresis.- Chromatography.- Amino Acid Analysis.- Protein Sequencing.- 6.3 Experimental Design and Procedures.- Preparation of the Acrylamide Gel.- Casting the Gel.- Assembling the Electrophoretic Buffer Chamber.- Sample Loading and Electrophoresis.- Coomassie Blue R-250 Staining.- Visualization by Assay withp-Nitrophenyl-?-D-galactoside.- Visualization by Fluorescence.- Study Questions.- Further Readings.- 7. Designing a Cloning Scheme.- 7.1 Overview.- 7.2 Background.- Biology of DNA.- Cloning Strategies.- Identifying Clones with Probes.- Probe Labelling Options.- 73 Experimental Design and Procedures.- Designing a Cloning Scheme.- Designing a Probe-DNA from Protein.- Preparation of Media for Yeast and E. col.- Streaking of Stored S. carlsbergensis and E. col.- Inoculation and Cultur-ing of Yeast and E. col.- Study Questions.- Further Readings.- 8. Isolation and Preparation of Nucleic Acids.- 8.1 Overview.- 8.2 Background.- Measuring Nucleic Acid Concentrations.- 8.3 Expermental Design and Procedures.- Yeast Genomic DNA Isolation.- Plasmid DNA Isolaion.- Estimation of DNA Concentrations.- Study Questions.- Further Readings.- 9. Constructing a Gene Bank.- 9.1 Overview.- 9.2 Background.- Cleaving DNA with Restriction Endonucleases.- Agarose Gel Electrophoresis.- Fragmenting Large Genomic DNA for Cloning.- The Ligation Reaction.- 9.3 Experimental Design and Procedures.- Cleaving Vector DNA with Restriction Endonucleases.- Partial Digestion of Genomic DNA.- Analysis of Restriction Digests by Agarose Gel Electrophoresis.- Ligation Reaction.- Study Questions.- Further Readings.- 10. Introducing Recombinant Molecules into Escherichia coli.- 10.1 Overview.- 10.2 Background.- Transformation Methodologies.- Selection of Transformed Organisms.- Identification of Recombinanat Vectors.- 10.3 Experimental Design and Procedures.- Preparation of Competent Cells.- Transformation of E. col.- Study Questions.- Further Readings.- 11. Screening for Clones.- 11.1 Overview.- 11.2 Background.- Parameters of Screening.- The Screening Process.- 11.3 Experimental Design and Procedures.- Tailing of an Oligonucleotide.- Synthesis of a Random Primed DNA Probe.- Testing of Digoxigenin Labelled Probes.- Colony Lifting and Probing of Transformed E. col.- Visualization of Colony Blots.- Picking and Culturing of Transformed Colonies.- Study Questions.- Further Readings.- 12. Characterizing and Verifying Cloned DNA.- 12.1 Overview.- 12.2 Background.- Restriction Endonuclease Mapping.- Southern Blotting.- DNA Sequencing.- Verifying the Clone.- 12.3 Experimental Design and Procedures.- Restriction Mapping.- Southern Blotting.- Colorimetric Detection.- Chemiluminescent Detection.- Evaluating the Southern Blot.- Sequencing of the MELI Gene.- Gel Preparation and Pouring.- Electrophoresing and Visualizing the Sequencing Reactions.- Transformation of Yeast with Cloned DNA.- Study Questions.- Further Readings.- 13. Application of DNA Sequence and Clone Data.- 13.1 Overview.- 13.2 Background.- Probe Design Using Molecular Analysis Software.- Basic Sequence Analysis.- Application of Sequence Data.- 13.3 Experimental Design and Procedures.- Computer Analysis of DNA Sequence.- Amplification of DNA by the Polymerase Chain Reaction.- Study Questions.- Further Readings.- 14. The Value of Information.