Biotechnology (eBook)

Front Cover 1
Calculations in Molecular Biology and Biotechnology 4
Copyright Page 5
Contents 8
Foreword 15
Chapter 1. Scientific Notation and Metric Prefixes 18
Introduction 18
Significant Digits 18
Rounding Off Significant Digits in Calculations 19
Exponents and Scientific Notation 21
Expressing Numbers in Scientific Notation 21
Converting Numbers from Scientific Notation to Decimal Notation 23
Adding and Subtracting Numbers Written in Scientific Notation 25
Multiplying and Dividing Numbers Written in Scientific Notation 26
Metric Prefixes 30
Conversion Factors and Canceling Terms 31
Chapter 2. Solutions Mixtures and Media 35
Introduction 35
Calculating Dilutions: A General Approach 35
Concentrations by a Factor of X 37
Preparing Percent Solutions 39
Diluting Percent Solutions 40
Moles and Molecular Weight: Definitions 44
Molarity 45
Diluting Molar Solutions 48
Converting Molarity to Percent 49
Converting Percent to Molarity 50
Normality 51
PH 52
pKa and the Henderson–Hasselbalch Equation 56
Chapter 3. Cell Growth 59
The Bacterial Growth Curve 59
Manipulating Cell Concentration 63
Plotting OD550 vs. Time on a Linear Graph 65
Plotting the Logarithm of OD550 vs. Time on a Linear Graph 66
Plotting the Log of Cell Concentration vs. Time 68
Calculating Generation Time 69
Plotting Cell Growth Data on a Semilog Graph 72
Determining Generation Time Directly from a Semilog Plot of Cell Concentration vs. Time 76
Plotting Cell Density versus OD550 on a Semilog Graph 77
The Fluctuation Test 78
Fluctuation Test Example 80
Variance 81
Measuring Mutation Rate 83
Measuring Cell Concentration on a Hemocytometer 92
Chapter 4. Working with Bacteriophage 94
Introduction 94
Multiplicity of Infection 94
Probabilities and Multiplicity of Infection 96
Measuring Phage Titer 102
Diluting Bacteriophage 103
Measuring Burst Size 104
Chapter 5. Quantitation of Nucleic Acids 107
Quantitation of Nucleic Acids by Ultraviolet Spectroscopy 107
Determining the Concentration of Double-Stranded DNA 108
Using Absorbance and an Extinction Coefficient to Calculate Double-Stranded DNA Concentration 111
Calculating DNA Concentration as a Millimolar (mM) Amount 113
Determining the Concentration of Single-Stranded DNA Molecules 114
Oligonucleotide Quantitation 116
Measuring RNA Concentration 120
Molecular Weight, Molarity, and Nucleic Acid Length 121
Estimating DNA Concentration on an Ethidium Bromide–Stained Gel 125
Chapter 6. Labeling Nucleic Acids with Radioisotopes 126
Introduction 126
Using Radioactivity: The Curie 126
Estimating Plasmid Copy Number 127
Labeling DNA by Nick Translation 129
Random Primer Labeling of DNA 131
Labeling 3' Termini with Terminal Transferase 136
cDNA Synthesis 138
Homopolymeric Tailing 145
In Vitro Transcription 150
Chapter 7. Oligonucleotide Synthesis 153
Introduction 153
Synthesis Yield 153
Measuring Stepwise and Overall Yield by the DMT Cation Assay 156
Overall Yield 156
Stepwise Yield 157
Calculating Micromoles of Nucleoside Added at Each Base Addition Step 159
Chapter 8. The Polymerase Chain Reaction 160
Introduction 160
Template and Amplification 160
Exponential Amplification 162
PCR Efficiency 164
Calculating the Tm of the Target Sequence 168
Primers 170
Primer Tm 175
dNTPs 182
DNA Polymerase 185
Quantitative PCR 188
Chapter 9. Recombinant DNA 203
Introduction 203
Restriction Endonucleases 203
The Frequency of Restriction Endonuclease Cut Sites 205
Calculating the Amount of Fragment Ends 206
Ligation 209
Transformation Efficiency 224
Genomic Libraries: How Many Clones Do You Need? 225
cDNA Libraries: How Many Clones Are Enough? 227
Expression Libraries 228
Screening Recombinant Libraries by Hybridization to DNA Probes 229
Sizing DNA Fragments by Gel Electrophoresis 241
Generating Nested Deletions Using Nuclease BAL 31 254
Chapter 10. Protein 259
Introduction 259
Protein Quantitation by Measuring Absorbance at 280 nm 259
Using Absorbance Coefficients and Extinction Coefficients to Estimate Protein Concentration 260
Relating Absorbance Coefficient to Molar Extinction Coefficient 262
Determining a Protein's Extinction Coefficient 263
Relating Concentration in Milligrams per Milliliter to Molarity 265
Protein Quantitation Using A280 When Contaminating Nucleic Acids Are Present 266
Protein Quantitation at 205 nm 267
Protein Quantitation at 205 nm When Contaminating Nucleic Acids Are Present 268
Measuring Protein Concentration by Colorimetric Assay- The Bradford Assay 269
Using ß-Galactosidase to Monitor Promoter Activity and Gene Expression 271
Specific Activity 274
The CAT Assay 277
Use of Luciferase in a Reporter Assay 282
In Vitro Translation–Determining Amino Acid Incorporation 283
Chapter 11. Centrifugation 287
Introduction 287
Relative Centrifugal Force (g Force) 287
Converting g Force to Revolutions per Minute 289
Determining g Force and Revolutions per Minute by Use of a Nomogram 290
Calculating Sedimentation Times 292
Chapter 12. Forensic Science 295
Introduction 295
Alleles and Genotypes 295
Calculating Genotype Frequencies 297
Calculating Allele Frequencies 298
The Hardy–Weinberg Equation and Calculating Expected Genotype Frequencies 299
The Chi-Square Test: Comparing Observed to Expected Values 303
Sample Variance 307
Sample Standard Deviation 308
Pi: The Power of Inclusion 309
Pd: The Power of Discrimination 310
DNA Typing and a Weighted Average 311
The Multiplication Rule 312
Index 314