Molecular Biology Techniques: A Classroom Laboratory Manual, Fifth Edition, is an in-depth laboratory manual for upper-level undergraduates and beginning graduate students in the general field of molecular biology techniques and biochemical applications. Presented as a series of coordinated experiments that mimics either a one- or two-semester research experience, the book also provides theories and explanations behind the techniques. Topics include manipulation of recombinant DNA, protein expression and purification, analysis of mRNA levels, mammalian cell culture techniques, CRISPR-mediated gene knockdown, and more. New topics, such as Gibson assembly experiments, have also been included. For instructors, the book contains easy to follow guides and preparation lists, designed to facilitate lab setup and pre-lab questions for use as a teaching tool.
1. Introduction - p. xix 2. Part I. Manipulation of DNA - p. 1 3. Advanced Alternatives Within Part I - p. 3 4. Lab Session 1. Getting Oriented; Practicing With Micropipettes - p. 7 5. Lab Session 2. Purification and Digestion of Plasmid (Vector) DNA - p. 13 6. Completion of Vector Preparation and Polymerase Chain Reaction Amplification of egfp - p. 23 7. Preparation of Insert DNA (egfp) PCR Product - p. 33 8. DNA Ligation and Transformation of Escherichia coli - p. 37 9. Part II. Screening Transformants - p. 43 10. Advanced Alternatives Within Part II - p. 45 11. Lab Session 6. Screening of Transformants, Part I - p. 47 12. Lab Session 7. Screening of Transformants, Part II - p. 53 13. Lab Session 8. Analysis of DNA Sequence From a Positive Clone, Part II - p. 63 14. Part III. Expression, Detection, and Purification of Recombinant Proteins From Bacteria - p. 71 15. Advanced Alternatives Within Part III - p. 73 16. Lab Session 9. Expression of Fusion Protein From Positive Clones, SDS–PAGE and Western Blot: Part I - p. 75 17. Lab Session 10. Expression of Fusion Protein From Positive Clones, SDS–PAGE, and Western Blot: Part II - p. 83 18. Extraction of Recombinant Protein From Escherichia coli Using a Glutathione Affinity Column - p. 87 19. Lab Session 12. Analysis of Purification Fractions - p. 95 20. Part IV. Analysis of mRNA Levels - p. 103 21. Lab Session 13. Total RNA Purification - p. 105 22. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part I - p. 113 23. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part II - p. 121 24. Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part I - p. 127 25. Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part II - p. 131 26. Part V. Modulation of Gene Expression - p. 135 27. Lab Session 18. Culturing Mammalian Cells - p. 137 28. Lab Session 19. Transient Transfection of Mammalian Cells - p. 143 29. Lab Session 20. RNAi-Mediated Knockdown of EGFP: Part I - p. 155 30. Lab Session 21. RNAi-Mediated Knockdown of EGFP: Part II - p. 161 31. Lab Session 22. RNAi-Mediated Knockdown of EGFP: Part III - p. 165 32. Lab Session 23. CRISPR-Mediated Knockout of EGFP: Part I - p. 169 33. Lab Session 24. CRISPR-Mediated Knockout of EGFP: Part II - p. 179 34. Lab Session 25. Advanced CRISPR: Part I - p. 181 35. Lab Session 26. Advanced CRISPR: Part II - p. 195 36. Lab Session 27. Interim Laboratory Session - p. 200 37. Lab Session 28. Advanced CRISPR: Part IV - p. 204 38. Lab Session 29. Advanced CRISPR: Part V - p. 209 39. Lab Session 30. Advanced CRISPR: Part VI - p. 213 40. Part VI. In Silico Tools - p. 219 41. Lab Session 31. Gibson Assembly In Silico Cloning - p. 221 42. Lab Session 32. Transcriptomic Analysis - p. 233 43. Lab Session 33. Structure-Activity Relationship Modeling - p. 239 44. Appendix A. Equipment - p. 249 45. Appendix B. Prep List - p. 251 46. Appendix C. Preparation of Competent Escherichia coli Cells - p. 281 47. Appendix D. Pre-Lab Questions - p. 283 48. Appendix E. Software Download Instructions for Lab Session 33: Structure-Activity Relationship Modeling - p. 301 49. Index - p. 305
Molecular Biology Techniques: A Classroom Laboratory Manual, Fifth Edition, is an in-depth laboratory manual for upper-level undergraduates and beginning graduate students in the general field of molecular biology techniques and biochemical applications. Presented as a series of coordinated experiments that mimics either a one- or two-semester research experience, the book also provides theories and explanations behind the techniques. Topics include manipulation of recombinant DNA, protein expression and purification, analysis of mRNA levels, mammalian cell culture techniques, CRISPR-mediated gene knockdown, and more. New topics, such as Gibson assembly experiments, have also been included. For instructors, the book contains easy to follow guides and preparation lists, designed to facilitate lab setup and pre-lab questions for use as a teaching tool.
1. Introduction - p. xix 2. Part I. Manipulation of DNA - p. 1 3. Advanced Alternatives Within Part I - p. 3 4. Lab Session 1. Getting Oriented; Practicing With Micropipettes - p. 7 5. Lab Session 2. Purification and Digestion of Plasmid (Vector) DNA - p. 13 6. Completion of Vector Preparation and Polymerase Chain Reaction Amplification of egfp - p. 23 7. Preparation of Insert DNA (egfp) PCR Product - p. 33 8. DNA Ligation and Transformation of Escherichia coli - p. 37 9. Part II. Screening Transformants - p. 43 10. Advanced Alternatives Within Part II - p. 45 11. Lab Session 6. Screening of Transformants, Part I - p. 47 12. Lab Session 7. Screening of Transformants, Part II - p. 53 13. Lab Session 8. Analysis of DNA Sequence From a Positive Clone, Part II - p. 63 14. Part III. Expression, Detection, and Purification of Recombinant Proteins From Bacteria - p. 71 15. Advanced Alternatives Within Part III - p. 73 16. Lab Session 9. Expression of Fusion Protein From Positive Clones, SDS–PAGE and Western Blot: Part I - p. 75 17. Lab Session 10. Expression of Fusion Protein From Positive Clones, SDS–PAGE, and Western Blot: Part II - p. 83 18. Extraction of Recombinant Protein From Escherichia coli Using a Glutathione Affinity Column - p. 87 19. Lab Session 12. Analysis of Purification Fractions - p. 95 20. Part IV. Analysis of mRNA Levels - p. 103 21. Lab Session 13. Total RNA Purification - p. 105 22. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part I - p. 113 23. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part II - p. 121 24. Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part I - p. 127 25. Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part II - p. 131 26. Part V. Modulation of Gene Expression - p. 135 27. Lab Session 18. Culturing Mammalian Cells - p. 137 28. Lab Session 19. Transient Transfection of Mammalian Cells - p. 143 29. Lab Session 20. RNAi-Mediated Knockdown of EGFP: Part I - p. 155 30. Lab Session 21. RNAi-Mediated Knockdown of EGFP: Part II - p. 161 31. Lab Session 22. RNAi-Mediated Knockdown of EGFP: Part III - p. 165 32. Lab Session 23. CRISPR-Mediated Knockout of EGFP: Part I - p. 169 33. Lab Session 24. CRISPR-Mediated Knockout of EGFP: Part II - p. 179 34. Lab Session 25. Advanced CRISPR: Part I - p. 181 35. Lab Session 26. Advanced CRISPR: Part II - p. 195 36. Lab Session 27. Interim Laboratory Session - p. 200 37. Lab Session 28. Advanced CRISPR: Part IV - p. 204 38. Lab Session 29. Advanced CRISPR: Part V - p. 209 39. Lab Session 30. Advanced CRISPR: Part VI - p. 213 40. Part VI. In Silico Tools - p. 219 41. Lab Session 31. Gibson Assembly In Silico Cloning - p. 221 42. Lab Session 32. Transcriptomic Analysis - p. 233 43. Lab Session 33. Structure-Activity Relationship Modeling - p. 239 44. Appendix A. Equipment - p. 249 45. Appendix B. Prep List - p. 251 46. Appendix C. Preparation of Competent Escherichia coli Cells - p. 281 47. Appendix D. Pre-Lab Questions - p. 283 48. Appendix E. Software Download Instructions for Lab Session 33: Structure-Activity Relationship Modeling - p. 301 49. Index - p. 305
