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Calculations for molecular biology and biotechnology / Frank Stephenson. -- 3rd ed. -- Boston, MA : Elsevier, c2016. – (58.178/S836/3rd ed.)

Contents

Biography  xiii

CHAPTER 1 Scientific Notation and Metric Prefixes  1

Introduction  1

1.1 Significant Digits  1

1.2 Exponents and Scientific Notation  3

1.3 Metric Prefixes  10

Chapter Summary  14

CHAPTER 2 Solutions, Mixtures, and Media  IS

Introduction  15

2.1 Calculating Dilutions: A General Approach  15

2.2 Concentrations by a Factor of X  17

2.3 Preparing Percent Solutions  19

2.4 Diluting Percent Solutions  20

2.5 Moles and Molecular Weight--Definitions  24

2.6 Normality  33

2.7 pH  34

2.8 pKa and The Henderson-Hasselbalch Equation  40

Chapter Summary  42

CHAPTER 3 Cell Growth  43

3.1  The Bacterial Growth Curve  43

3.2  Manipulating Cell Concentration  48

3.3  Plotting OD550 Versus Time on a Linear Graph  51

3.4  Plotting the Logarithm of OD550 Versus Time on a Linear Graph  52

3.5  Plotting the Log of Cell Concentration Versus Time  54

3.6  Calculating Generation Time  55

3.7  Plotting Cell Growth Data on a Semilog Graph  57

3.8  Plotting Cell Concentration Versus Time on a Semilog Graph  60

3.9  Determining Generation Time Directly From a Semilog Plot of Cell Concentration Versus Time  61

3.10 Plotting Cell Density Versus ODsso on a Semilog Graph  62

3.11 The Fluctuation Test  63

3.12 Measuring Mutation Rate  68

3.13 Measuring Cell Concentration on a Hemocytometer  77

Chapter Summary  78

Reference  79

CHAPTER 4 Working with Bacteriophage  81

Introduction  81

4.1 Multiplicity of Infection  81

4.2 Probabilities and Multiplicity of infection  83

4.3 Measuring Phage Titer  89

4.4 Diluting Bacteriophage  91

4.5 Measuring Burst Size  93

Chapter Summary  96

CHAPTER 5 Nucleic Acid 0uantifioti0n  97

5.1 Quantification of Nucleic Acids by Ultraviolet Spectroscopy  97

5.2 Determining the Concentration of Double-Stranded DNA  98

5.3 Determining the Concentration of Single-Stranded DNA Molecules  107

5.4 0ligonucleotide Quantification  109

5.5 Measuring RNA Concentration  114

5.6 Molecular Weight, Molarity, and Nucleic Acid Length  114

5.7 Estimating DNA Concentration on an Ethidium Bromide-Stained Gel  120

5.8 Dye-Labeled Nucleic Acids  121

5.9 Limit of Detection and Limit of Quantitation  124

Chapter Summary  128

CHAPTER 6 Labeling Nucleic Acids With Radioisotopes  131

Introduction  131

6.1 Units of Radioactivity: The Curie  131

6.2 Estimating Plasmid Copy Number  132

6.3 Labeling DNA by Nick Translation  134

6.4 Random Primer Labeling of DNA  136

6.5 Labeling 3’Termini With Terminal Transferase  141

6.6 Complementary DNA Synthesis  143

6.7 Homopolymeric Tailing  149

6.8 In Vitro Transcription  155

Chapter Summary  157

CHAPTER 7 Oligonucleotide Synthesis  161

Introduction  161

7.1 Synthesis Yield  162

7.2 Measuring Stepwise and Overall Yield by the Dimethoxytrityl Cation Assay  164

7.3 Calculating Micromoles of Nucleoside Added at Each Base Addition Step  167

Chapter Summary  168

CHAPTER 8 The Polymerase Chain Reaction  171

Introduction  171

8.1 Template and Amplification  171

8.2 Exponential Amplification  173

8.3 Polymerase Chain Reaction Efficiency  176

8.4 Calculating the Tm of the Target Sequence  ~  179

8.5 Primers  182

8.6 Primer Tm  187

8.7 Deoxynucleoside Triphosphates  194

8.8 DNA Polymerase  196

8.9 Quantitative PCR  199

Chapter Summary  211

References  213

Further Reading  213

CHAPTER 9 Real-Time PCR  215

Introduction  215

9.1  The Phases of a Real-Time Polymerase Chain Reaction  216

9.2  Controls  218

9.3  Absolute Quantification by the TaqMan Assay  220

9.4  Amplification Efficiency  235

9.5  Measuring Gene Expression  239

9.6  Relative Quantification: The AA, CT Method  241

9.7  Relative Standard Curve Method  276

9.8  Relative Quantification by Reaction Kinetics  294

9.9  The R0 Method of Relative Quantification  300

9.10 The Pfaffl Model  303

9.11 Digital Polymerase Chain Reaction  306

Chapter Summary  315

References  319

Further Reading  320

CHAPTER 10 Recombinant DNA  321

introduction  321

10.1 Restriction Endonucleases  321

10.2 Calculating the Amount of Fragment Ends  324

10.3 Ligation  327

10.4 Genomic Libraries--How Many Clones Do You Need?  343

10.5 cDNA Libraries--How Many Clones Are Enough?  344

10.6 Expression Libraries  345

10.7 Screening Recombinant Libraries by Hybridization to DNA Probes  347

10.8 Sizing DNA Fragments by Gel Electrophoresis  357

10.9 Generating Nested Deletions Using Nuclease BAL 31  365

Chapter Summary  369

References  373

CHAPTER 11 Protein  375

Introduction  375

11.1  Calculating a Protein's Molecular Weight From Its Sequence

11.2  Determining a Protein's Molecular Weight by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis  379

11.3  Protein Quantification by Measuring Absorbance at 280 nm  382

11.4  Using Absorbance Coefficients and Extinction Coefficients to Estimate Protein Concentration  383

11.5  Relating Concentration in Milligrams per Milliliter to Molarity  390

11.6  Protein Quantitation Using A280 When Contaminating Nucleic Acids Are Present

11.7  Protein Quantification at 205 nm  392

11.8  Protein Quantitation at 205 nm When Contaminating Nucleic Acids Are Present  393

11.9  Labeling Proteins With Fluorescent Dyes  394

11.10 Measuring Protein Concentration by Colorimetric Assay--The Bradford Assay  397

11.11 Thin-Layer Chromatography and Rf  399

11.12 Estimating a Protein's Molecular Weight by Gel Filtration  401

11.13 Protein Volume  406

11.14 Using 13-Galactosidase to Monitor Promoter Activity and Gene Expression  408

11.15 The Chloramphenicol Acetyltransferase Assay  412

11.16 Use of Luciferase in a Reporter Assay  416

11.17 DNA Polymerase Activity  417

11.18 In Vitro Translation--Determining Amino Acid Incorporation  421

11.19 The Isoelectric Point of a Protein  422

Chapter Summary  425

References  428

Further Reading  429

CHAPTER 12 Centrifugation  431

Introduction  431

12.1 Relative Centrifugal Force (g Force)  431

12.2 Calculating Sedimentation Times  436

Chapter Summary  438

Further Reading  438

CHAPTER 13 Forensics and Paternity  439

Introduction  439

13.1 Alleles and Genotypes  440

13.2 The Hardy-Weinberg Equation and Calculating Expected Genotype Frequencies  443

13.3 The Chi-Square Test--Comparing Observed to Expected Values  446

13.4 The Power of Inclusion  451

13.5 The Power of Discrimination  452

13.6 DNA Typing and a Weighted Average  453

13.7 The Multiplication Rule  454

13.8 The Paternity Index  454

Chapter Summary  459

More Problems  460

References  462

Further Reading  462

Appendix A: Using Microsoft Excel's Graphing Utility  465

Index  477