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.

This text is broken into three sections based on level Methods in Biotechnology, Advanced Methods in Biotechnology I, and Advanced Methods in Biotechnology II. Each section contains 14–22 lab exercises, with instructor notes in appendices as well as an answer guide as a part of the book companion site. This text will be an excellent resource for both students and laboratory professionals in the biotechnology field.

 Uniquely implements a holistic approach to biotechnology that lends itself well to use for both instruction and research in advanced biotechnology laboratories
 Provides a scheduled framework for students learning advanced laboratory techniques and  how to design experiments and interpret results
 Includes basic biochemical techniques and bioinformatics exercises,  as well as laboratory mathematics practice problem sets (with answer keys)
 Encourages students to engage in a higher level of critical thinking and troubleshooting, rather than simply performing experiments
 Includes access to a companion website hosting book content including figures, tables, etc.

About the Authors
Seung–Beom Hong is a Visiting Assistant Professor of Biotechnology at the University of Houston–Clear Lake in Houston, 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 –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