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