Molecular Biology Techniques: A Classroom Laboratory Manual

Molecular Biology Techniques: A Classroom Laboratory Manual, Fourth Edition is a must-have collection of methods and procedures on how to create a single, continuous, comprehensive project that teaches students basic molecular techniques. It is 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 will gain hands-on experience on subcloning a gene into an expression vector straight through to the purification of the recombinant protein.

Presents student-tested labs proven successful in real classroom laboratoriesIncludes a test bank on a companion website for additional testing and practiceProvides exercises that simulate a cloning project that would be performed in a real research labIncludes a prep-list appendix that contains necessary recipes and catalog numbers, providing staff with detailed instructions

Part I: Manipulation of DNA Advanced Alternatives Within Part I 1. Getting Oriented; Practicing With Micropipettes 2. Purification and Digestion of Plasmid (Vector) DNA 3. Completion of Vector Preparation and Polymerase Chain Reaction Amplification of egfp 4. Preparation of Insert DNA (egfp) PCR Product 5. DNA Ligation and Transformation of Escherichia coli

Part II: Screening Transformants Advanced Alternatives Within Part II 6. Screening of Transformants, Part I 7. Screening of Transformants, Part II 8. Analysis of DNA Sequence From a Positive Clone, Part II

Part III: Expression, Detection, and Purification of Recombinant Proteins from Bacteria Advanced Alternatives Within Part III 9. Expression of Fusion Protein from Positive Clones, SDS_PAGE and Western Blot: Part I 10. Expression of Fusion Protein from Positive Clones, SDS_PAGE, and Western Blot: Part II 11. Extraction of Recombinant Protein From Escherichia coli Using a Glutathione Affinity Column 12. Analysis of Purification Fractions

Part IV: Analysis of mRNA Levels 13. Total RNA Purification 14. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part I 15. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part II 16. Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part I 17. Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part II

Part V: Modulation of Gene Expression 18. Culturing Mammalian Cells 19. Transient Transfection of Mammalian Cells 20. RNAi-Mediated Knockdown of EGFP: Part I 21. RNAi-Mediated Knockdown of EGFP: Part II 22. RNAi-Mediated Knockdown of EGFP: Part III 23. CRISPR-Mediated Knockout of EGFP: Part I 24. CRISPR-Mediated Knockout of EGFP: Part II 25. Advanced CRISPR: Part I 26. Advanced CRISPR: Part II 27. Interim Laboratory Session 28. Advanced CRISPR: Part IV 29. Advanced CRISPR: Part V 30. Advanced CRISPR: Part VI

Appendix A: Equipment B: Prep List  C: Preparation of Competent Escherichia coli Cells  D: Pre-Lab Questions