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Full Description
Molecular Biology TechniquesEdition is a must-have collection of methods and procedures that create a single, continuous, comprehensive project that teaches students basic molecular techniques. It is an an indispensable tool for introducing advanced undergraduates and beginning graduate students to the techniques of recombinant DNA technology, or gene cloning and expression. The techniques used in basic research and biotechnology laboratories are covered in detail. Students gain hands-on experience from start to finish in subcloning a gene into an expression vector, through purification of the recombinant protein.The fourth edition is updated with new laboratory exercises, designed for a typical 15-week semester, rather than a 4-week intensive course. The "project" approach to experiments has been student and instructor tested for years: students still follow a cloning project through to completion, culminating in the purification of recombinant protein. It takes advantage of the enhanced green fluorescent protein - students can actually visualize positive clones following IPTG induction.
Contents
Part 1. Manipulation of DNA Lab Session 1. Getting Oriented Lab Session 2. Purification and Digestion of Plasmid (Vector) DNA Lab Session 3. PCR Amplification of egfp and Completion of Vector Preparation Lab Session 4. Preparation of Insert DNA (egfp) PCR Product Lab Session 5. DNA Ligation and Transformation of Escherichia coliPart 2. Screening Transformants Lab Session 6. Colony Hybridization Lab Session 6A. Interim Laboratory Session Lab Session 6B. Colony Hybridization: Monoclonal Antibody Probe Lab Session 7. Characterization of Recombinant Clones Lab Session 7A. Completion of Colony Hybridization with a Monoclonal Antibody Probe Lab Session 7B. PCR Screening Lab Session 7C. Prepare Fresh Replica Plate Lab Session 8. Characterization of Recombinant Clones Lab Session 8A. Interim Laboratory Session Lab Session 8B. Analysis of PCR Screen Results Lab Session 8C. Isolation of Miniprep DNA from Potential Transformants Lab Session 8D. Visualization of Green Fluorescent Protein: Part 1 Lab Session 9. Characterization of Recombinant Clones Lab Session 9A. Characterization of Miniprep DNA from Potential Transformants (Restriction Enzyme Analysis of Putative Transformants) Lab Session 9B. Visualization of Green Fluorescent Protein: Part 2 Lab Session 9C. Computational Analysis of DNA Sequence from a Positive Clone: Part 1 Lab Session 10. Computational Analysis of DNA Sequence from a Positive ClonePart 3. Expression, Detection and Purification of Recombinant Proteins from Bacteria Lab Session 11. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot Lab Session 11A. Interim Laboratory Session Lab Session 11B. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot Lab Session 12. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot Lab Session 13. Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column Lab Session 13A. Interim Laboratory Session Lab Session 13B. Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column Lab Session 14. Analysis of Purification Fractions Lab Session 14A. Analysis of Purification Fractions Lab Session 14B. Replica Plate Positive Clone Part 4. Analysis of mRNA Levels Lab Session 15. Total RNA Purification Lab Session 15A. Interim Laboratory Session Lab Session 15B. Total RNA Purification Lab Session 16. Analysis of gst::egfp mRNA Levels by RT-qPCR Lab Session 17. Analysis of gst::egfp mRNA Levels by RT-qPCR Lab Session 18. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR Lab Session 19. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCRAppendix 1. Equipment 2. Prep List 3. Preparation of Competent E. coli Cells 4. Pre-Lab Questions
