Molecular Biology Techniques. A Classroom Laboratory Manual. Edition No. 4

Table of Contents

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

Authors

Sue Carson North Carolina State University, Raleigh, U.S.A.. Heather B. Miller Department of Chemistry, High Point University, High Point, NC USA. Dr. Heather Miller is an Assistant Professor of Biochemistry in the Chemistry Department at High Point University (High Point, NC). She graduated from Clarion University of Pennsylvania (Clarion, PA) with a B.S. in Molecular Biology/Biotechnology, and from Duke University (Durham, NC) with a Ph.D. in Molecular Genetics and Microbiology. She completed a teaching postdoctoral position in the Biotechnology Program at North Carolina State University (Raleigh, NC). Her area of scientific expertise is RNA biology. Her research focuses on HIV-1 gene expression and the coupling of transcription and splicing in humans. She has taught at the college level for nine years, and is engaged in the scholarship of teaching and learning. Melissa C. Srougi Assistant Professor of Biochemistry, High Point University. Dr. Melissa C. Srougi is an Assistant Professor of Biochemistry at High Point University. She graduated from the University of Toledo (Toledo, OH) with a B.S. in Biology, and from Case Western Reserve University (Cleveland, OH) with a Ph.D. in Pharmacology. Her scientific areas of expertise are in experimental cancer chemotherapeutic agents and mechanisms of chemotherapeutic resistance. Dr. Srougi actively trains undergraduate research students in her laboratory. In addition, she has developed and taught a variety of inquiry-based college level science courses and has published and presented a number of peer-reviewed papers in the scholarship of teaching and learning. D. Scott Witherow Department of Chemistry, Biochemistry, and Physics, The University of Tampa, Tampa, FL USA. Dr. Witherow graduated from Rollins College (Winter Park, FL) with an A.B. in Chemistry, and from the University of Miami (Miami, FL) with a Ph.D. in Molecular and Cellular Pharmacology. His research has focused primarily on G protein-mediated signal transduction processes in mammalian systems. Following two research postdoctoral fellowships, Dr. Witherow served as a teaching postdoctoral fellow at North Carolina State University, where he published and presented multiple papers in the field of science education and developed a passion for teaching. He is currently an Associate Professor at The University of Tampa, where he has been teaching undergraduate students since 2011.