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Calculations for molecular biology and biotechnology : A guide to mathematics in the laboratory / Frank H. Stephenson. — 2nd ed. — Amsterdam : Elsevier Academic Press, 2010. – (58.178/S836/2nd ed.) |
Contents
Contents
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 15
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 34
2.7 pH 35
2.8 pKa and the Henderson-Hasselbalch Equation 40
Chapter Summary 43
CHAPTER 3 Cell Growth 45
3.1 The Bacteria] Growth Curve 45
3.2 Manipulating Cell Concentration
3.3 Plotting OD550 vs. Time on a Linear Graph
3.4 Plotting the Logarithm of OD550 vs. Time on a Linear Graph
3.5 Plotting the Logarithm of Cell Concentration vs. Time
3.6 Calculating Generation Time
3.7 Plotting Cell Growth Data on a Semilog Graph
3.8 Plotting Cell Concentration vs. Time on a Semilog Graph
3.9 Determining Generation Time Directly from a Semilog Plot of Cell Concentration vs. Time
3.10 Plotting Cell Density vs. OD550 on a Semilog Graph
3.11 The Fluctuation Test
3.12 Measuring Mutation Rate
3.13 Measuring Cell Concentration on a Hemocytometer
Chapter Summary
References
CHAPTER 4 Working with Bacteriophages 83
Introduction 83
4.1 Multiplicity of Infection (moi) 83
4.2 Probabilities and Multiplicity of Infection (moi) 85
4.3 Measuring Phage Titer 91
4.4 Diluting Bacteriophage 93
4.5 Measuring Burst Size 95
Chapter Summary 98
CHAPTER 5 Nucleic Acid Quantification 99
5.1 Quantification of Nucleic Acids by Ultraviolet (UV) Spectroscopy
5.2 Determining the Concentration of Double-Stranded DNA (dsDNA)
5.3 Determining the Concentration of Single-Stranded DNA (ssDNA) Molecules 108
5.4 Oligonucleotide Quantification 111
5.5 Measuring RNA Concentration 115
5.6 Molecular Weight， Molarity， and Nucleic Acid Length 115
5.7 Estimating DNA Concentration on an Ethidium Bromide-Stained Gel
Chapter Summary 121
CHAPTER 6 Labeling Nucleic Acids with Radioisotopes 123
Introduction 123
6.1 Units of Radioactivity - The Curie (Ci) 123
6.2 Estimating Plasmid Copy Number 124
6.3 Labeling DNA by Nick Translation 126
6.4 Random Primer Labeling of DNA 128
6.5 Labeling 3' Termini with Terminal Transferase 133
6.6 Complementary DNA (cDNA) Synthesis 135
6.7 Homopolymeric Tailing 141
6.8 In Vitro Transcription
Chapter Summary
CHAPTER 7 0ligonucleotide Synthesis 155
Introduction 155
7.1 Synthesis Yield 156
7.2 Measuring Stepwise and Overall Yield by the Dimethoxytrityl (DMT) Cation Assay
7.3 Calculating Micromoles of Nucleoside Added at Each Base Addition Step 161
Chapter Summary 162
CHAPTER 8 The Polymerase Chain Reaction (PCR) 165
Introduction 165
8.1 Template and Amplification 165
8.2 Exponential Amplification 167
8.3 Polymerase Chain Reaction (PCR) Efficiency 170
8.4 Calculating the T，， of the Target Sequence 173
8.5 Primers 176
8.6 Primer Tm 181
8.7 Deoxynucleoside Triphosphates (dNTPs) 189
8.8 DNA Polymerase 191
8.9 Quantitative Polymerase Chain Reaction (PCR) 195
Chapter Summary
References
Further Reading
CHAPTER 9 The Real-time Polymerase Chain Reaction (RT-PCR) 211
Introduction 211
9.1 The Phases of Real-time PCR 212
9.2 Controls 215
9.3 Absolute Quantification by the TaqMan Assay 216
9.4 Amplification Efficiency 232
9.5 Measuring Gene Expression 236
9.6 Relative Quantification - The AACT Method 238
9.7 The Relative Standard Curve Method
9.8 Relative Quantification by Reaction Kinetics.
9.9 The Ro Method of Relative Quantification ...
9.10 The Pfaffl Model
Chapter Summary
References
Further Reading
CHAPTER 10 Recombinant DNA 313
Introduction 313
10.1 Restriction Endonucleases 313
10.2 Calculating the Amount of Fragment Ends 316
10.3 Ligation 319
10.4 Genomic Libraries - How Many Clones Do You Need? 336
10.5 cDNA Libraries - How Many Clones are Enough? 337
10.6 Expression Libraries 339
10.7 Screening Recombinant Libraries by Hybridization to DNA Probes
10.8 Sizing DNA Fragments by Gel Electrophoresis 351
10.9 Generating Nested Deletions Using Nuclease BAL 31 359
Chapter Summary 363
References 367
CHAPTER 11 Protein 369
Introduction 369
11.1 Calculating a Protein's Molecular Weight from Its Sequence
11.2 Protein Quantification by Measuring Absorbance at 280 nm 373
11.3 Using Absorbance Coefficients and Extinction Coefficients to Estimate Protein Concentration 374
11.4 Relating Concentration in Milligrams Per Milliliter to Molarity 380
11.5 Protein Quantitation Using A280 When Contaminating Nucleic Acids are Present
11.6 Protein Quantification at 205 nm 383
11.7 Protein Quantitation at 205 nm When Contaminating Nucleic Acids are Present
11.8 Measuring Protein Concentration by Colorimetric Assay - The Bradford Assay
11.9 Using-Galactosidase to Monitor Promoter Activity and Gene Expression 387
11.10 Thin Layer Chromatography (TLC) and the Retention Factor (Rf)
11.11 Estimating a Protein's Molecular Weight by Gel Filtration 394
11.12 The Chloramphenicol Acetyltransferase (CAT) Assay 399
11.13 Use of Luciferase in a Reporter Assay 403
11.14 In Vitro Translation - Determining Amino Acid Incorporation
11.15 The Isoelectric Point (pI) of a Protein 405
Chapter Summary 408
References 41l
Further Reading 412
CHAPTER 12 Centrifugation 413
Introduction 413
12.1 Relative Centrifugal Force (RCF) (g Force) 413
12.2 Calculating Sedimentation Times 418
Chapter Summary 420
References 421
Further Reading 421
CHAPTER 13 Forensics and Paternity 423
Introduction 423
13.1 Alleles and Genotypes 424
13.2 The Hardy-Weinberg Equation and Calculating Expected Genotype Frequencies
13.3 The Chi-Square Test - Comparing Observed to Expected Values
13.4 The Power of Inclusion (Pi)
13.5 The Power of Discrimination (Pd)
13.6 DNA Typing and Weighted Average
13.7 The Multiplication Rule
13.8 The Paternity Index (PI)
Chapter Summary
References
Further Reading
Appendix A
Index
