Biotechnology Proteins to PCR: A Course in Strategies and Lab Techniques

ISBN-13: 9780817638436 / Angielski / Miękka / 1995 / 317 str.

D. Burden; Donald B. Whitney; David W. Burden

Opis

ism (i. e., Saccharomyces carlsbergensis, or brewer's yeast) and one of its corresponding enzymes. The experiments on this organism and enzyme are not limited to the materials suggested and can be easily adapted to the desired technical level and available budget. Similarly, the subse quent cloning experiments suggest that use of particular vectors and strains, but, as indicated, alternative materials can be used to success fully perform the laboratory exercises. We would like to thank the corporate sponsors of the Biotechnology Training Institute for providing the materials and expertise for the devel opment of our programs, and thus for the materials in this manual. These sponsors include: Barnsteadffhermolyne, Dubuque, IA Beckman Instruments, Somerset, NJ Bio-Rad Laboratories, Hercules, CA Boehringer Mannheim Corporation, Indianapolis, IN Coming Costar Corporation, Cambridge, MA FMC BioProducts, Rockland, ME Kodak Laboratory Products, New Haven, CT Labconco, Kansas City, MO MJ Research, Cambridge, MA Olympus Instruments, Lake Success, NY Pharmacia Biotech, Piscataway, NJ Savant, Inc., Farmingdale, NY VWR Scientific, Philadelphia, P A We would also like to thank the following individuals for their input, comments, and suggestions: Tom Slyker, Bernie Janoson, Steven Piccoli, John Ford, JeffGarelik, Yanan Tian, and Douglas Beecher. Special thanks to Alan Williams for his critique of the chromatography experiments and Shannon Gentile for her work in the laboratory. We would especial ly like to thank Maryann Burden for her comments and encouragement.

Spis treści

1. 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.