Methods in Biotechnology

As rapid advances in biotechnology occur, there is a need for a pedagogical tool to aid current students and laboratory professionals in biotechnological methods; Methods in Biotechnology is an invaluable resource for those students and professionals.

Methods in Biotechnology engages the reader by implementing an active learning approach, provided advanced study questions, as well as pre- and post-lab questions for each lab protocol. These self-directed study sections encourage the reader to not just perform experiments but to engage with the material on a higher level, utilizing critical thinking and troubleshooting skills.

SEUNG-BEOM HONG is a Visiting Assistant Professor of Biotechnology at the University of Houston-Clear Lake in Houston, TX, USA.

M. BAZLUR RASHID is an Associate Professor of Biology and Biotechnology and former Program Chair of Biotechnology at the University of Houston-Clear Lake in Houston, TX, USA.

LORY Z. SANTIAGO-VÁZQUEZ is an Associate Professor of Biotechnology at the University of Houston-Clear Lake in Houston, TX, USA.

Preface vii

Acknowledgements ix

Introduction to biotechnology lab xi

About the companion website xix

Part I: Methods in Biotechnology (MB) Laboratory Exercises 1

1 MB experiment 1: Lab measurements 3

2 MB experiment 2: Use of the spectrophotometer and Beer’s law 9

3 MB experiment 3: Making solutions and buffer efficacy 15

4 MB experiment 4: Acid–base titration 19

5 MB experiment 5: Protein denaturation and precipitation 23

6 MB experiment 6: Bacterial transformation 27

7 MB experiment 7: GFP purification 33

8 MB experiment 8: SDS-PAGE analysis 37

9 MB experiment 9: DNA isolation 41

10 MB experiment 10: PCR-based Alu-human DNA typing 45

11 MB experiment 11: Restriction enzyme digestion 51

12 MB experiment 12: Agarose gel electrophoresis 53

13 MB experiment 13: Ouchterlony and ELISA immunoassays 57

14 MB experiment 14: Testing plant substances for antimicrobial activity 63

15 MB experiment 15: Peroxidase enzyme activity assay 67

Part II: Advanced Methods in Biotechnology (AMB) 1 Laboratory Exercises 71

16 AMB 1 experiment 16: Aseptic technique and culture handling 73

17 AMB 1 experiment 17: Yeast culture media preparation 77

18 AMB 1 experiment 18: Growth curve 79

19 AMB 1 experiment 19: Mini plasmid prep 83

20 AMB 1 experiment 20: Restriction digestion, purification concentration and quantification of DNA 87

21 AMB 1 experiment 21: Polymerase chain reaction (PCR) 91

22 AMB 1 experiment 22: TA blunt end SLIC, and CPEC cloning of PCR product 97

23 AMB 1 experiment 23: One-step multifragment assembly cloning 103

24 AMB 1 experiment 24: Restriction enzyme digestion and fast agarose gel electrophoresis 111

25 AMB 1 experiment 25: Southern blot transfer 117

26 AMB 1 experiment 26: Probe labeling and purification 121

27 AMB 1 experiment 27: Dot blot assay 125

28 AMB 1 experiment 28: Pre-hybridization, hybridization and detection 127

29 AMB 1 experiment 29: Total yeast RNA isolation and RT-PCR 131

30 AMB 1 experiment 30: Yeast-based in vivo recombination cloning 139

31 AMB 1 experiment 31: Plasmid DNA isolation from yeast 143

32 AMB 1 experiment 32: E. coli transformation with yeast plasmid DNA 145

33 AMB 1 experiment 33: X-gal filter lift assay 147

34 AMB 1 experiment 34: Protein quantitation assay 149

35 AMB 1 experiment 35: Quantitative 𝛃-Galactosidase assay in yeast 155

36 AMB 1 experiment 36: Gel filtration chromatography (GFC) 161

37 AMB 1 experiment 37: Ion exchange chromatography (IEC) 165

Part III: Advanced Methods in Biotechnology (AMB) 2 Laboratory Exercises 169

38 AMB 2 experiment 38: E. coli culture media preparation 171

39 AMB 2 experiment 39: Site-directed mutagenesis 175

40 AMB 2 experiment 40: Protein expression in E. coli 179

41 AMB 2 experiment 41: Protein purification by affinity column chromatography 187

42 AMB 2 experiment 42: SDS-PAGE analysis of affinity column fractions 193

43 AMB 2 experiment 43: Western blot analysis of affinity column fractions 199

44 AMB 2 experiment 44: Yeast media preparation and phenotypic analysis of yeast strains 203

45 AMB 2 experiment 45: Yeast transformation for yeast two-hybrid (Y2H) assay and genome editing by CRISPR-Cas system 209

46 AMB 2 experiment 46: Yeast mating-mediated Y2H assay and genomic PCR 215

47 AMB 2 experiment 47: Yeast colony PCR screening and cycle DNA sequencing 221

48 AMB 2 experiment 48: DNA sequencing electrophoresis 227

49 AMB 2 experiment 49: RNA interference 237

50 AMB 2 experiment 50: Protein preparation for 2D gel electrophoresis 241

51 AMB 2 experiment 51: Two-dimensional gel electrophoresis 245

Part IV: Appendices 253

1 Methods in Biotechnology Appendix 1 255

2 MB Appendix 2 261

3 MB Appendix 3 265

4 MB Appendix 4 267

5 AMB 1 Appendix 1 271

6 AMB 1 Appendix 2 281

7 AMB 1 Appendix 3 287

8 AMB 1 Appendix 4 291

9 AMB 1 Appendix 5 297

10 AMB 2 Appendix 1 299

11 AMB 2 Appendix 2 307

12 AMB 2 Appendix 3 313

13 AMB 2 Appendix 4 317

14 AMB 2 Appendix 5 319

15 AMB 2 Appendix 6 321

16 AMB 2 Appendix 7 325

Glossary 327

Abbreviations 335

Index 337

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