Molecular biology techniques : a classroom laboratory manual / by Susan Carson ... [et al.]. -- 4th ed. -- London ; Academic Press, 2019. – (58.21 /R649 /4th ed.)

Contents

About the Authors

Preface

Acknowledgements

Note to Instructors

Nomenclature

Introduction

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 coil

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     95

Part IV Analysis of mRNA levels

13. Total RNA Purification             105

14. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part I                113

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

Appendix B: Prep List

Appendix C: Preparation of Competent Escherichia coli Cells

Appendix D: Pre-Lab Questions

Index