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Calculations for Molecular Biology and Biotechnology - A Guide to Mathematics in the Laboratory 2e
Front Cover
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Calculations for Molecular Biology and Biotechnology
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Copyright Page
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Contents
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CHAPTER 1 Scientific Notation and Metric Prefixes
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Introduction
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1.1 Significant Digits
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1.1.1 Rounding Off Significant Digits in Calculations
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1.2 Exponents and Scientific Notation
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1.2.1 Expressing Numbers in Scientific Notation
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1.2.2 Converting Numbers from Scientific Notation to Decimal Notation
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1.2.3 Adding and Subtracting Numbers Written in Scientific Notation
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1.2.4 Multiplying and Dividing Numbers Written in Scientific Notation
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1.3 Metric Prefixes
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1.3.1 Conversion Factors and Canceling Terms
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Chapter Summary
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CHAPTER 2 Solutions, Mixtures, and Media
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Introduction
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2.1 Calculating Dilutions – A General Approach
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2.2 Concentrations by a Factor of X
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2.3 Preparing Percent Solutions
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2.4 Diluting Percent Solutions
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2.5 Moles and Molecular Weight – Definitions
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2.5.1 Molarity
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2.5.2 Preparing Molar Solutions in Water with Hydrated Compounds
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2.5.3 Diluting Molar Solutions
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2.5.4 Converting Molarity to Percent
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2.5.5 Converting Percent to Molarity
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2.6 Normality
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2.7 pH
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2.8 pK[sub(a)] and the Henderson–Hasselbalch Equation
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Chapter Summary
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CHAPTER 3 Cell Growth
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3.1 The Bacterial Growth Curve
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3.1.1 Sample Data
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3.2 Manipulating Cell Concentration
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3.3 Plotting OD[sub(550)] vs. Time on a Linear Graph
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3.4 Plotting the Logarithm of OD[sub(550)] vs. Time on a Linear Graph
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3.4.1 Logarithms
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3.4.2 Sample OD[sub(550)] Data Converted to Logarithm Values
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3.4.3 Plotting Logarithm OD[sub(550)] vs. Time
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3.5 Plotting the Logarithm of Cell Concentration vs. Time
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3.5.1 Determining Logarithm Values
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3.6 Calculating Generation Time
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3.6.1 Slope and the Growth Constant
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3.6.2 Generation Time
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3.7 Plotting Cell Growth Data on a Semilog Graph
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3.7.1 Plotting OD[sub(550)] vs. Time on a Semilog Graph
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3.7.2 Estimating Generation Time from a Semilog Plot of OD[sub(550)] vs. Time
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3.8 Plotting Cell Concentration vs. Time on a Semilog Graph
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3.9 Determining Generation Time Directly from a Semilog Plot of Cell Concentration vs. Time
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3.10 Plotting Cell Density vs. OD[sub(550)] on a Semilog Graph
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3.11 The Fluctuation Test
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3.11.1 Fluctuation Test Example
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3.11.2 Variance
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3.12 Measuring Mutation Rate
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3.12.1 The Poisson Distribution
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3.12.2 Calculating Mutation Rate Using the Poisson Distribution
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3.12.3 Using a Graphical Approach to Calculate Mutation Rate from Fluctuation Test Data
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3.12.4 Mutation Rate Determined by Plate Spreading
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3.13 Measuring Cell Concentration on a Hemocytometer
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Chapter Summary
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References
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CHAPTER 4 Working with Bacteriophages
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Introduction
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4.1 Multiplicity of Infection (moi)
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4.2 Probabilities and Multiplicity of Infection (moi)
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4.3 Measuring Phage Titer
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4.4 Diluting Bacteriophage
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4.5 Measuring Burst Size
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Chapter Summary
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CHAPTER 5 Nucleic Acid Quantification
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5.1 Quantification of Nucleic Acids by Ultraviolet (UV) Spectroscopy
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5.2 Determining the Concentration of Double-Stranded DNA (dsDNA)
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5.2.1 Using Absorbance and an Extinction Coefficient to Calculate Double-Stranded DNA (dsDNA) Concentration
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5.2.2 Calculating DNA Concentration as a Millimolar (mM) Amount
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5.2.3 Using PicoGreen® to Determine DNA Concentration
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5.3 Determining the Concentration of Single-Stranded DNA (ssDNA) Molecules
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5.3.1 Single-Stranded DNA (ssDNA) Concentration Expressed in μg/mL
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5.3.2 Determining the Concentration of High-Molecular-Weight Single-Stranded DNA (ssDNA) in pmol/μL
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5.3.3 Expressing Single-Stranded DNA (ssDNA) Concentration as a Millimolar (mM) Amount
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5.4 Oligonucleotide Quantification
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5.4.1 Optical Density (OD) Units
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5.4.2 Expressing an Oligonucleotide's Concentration in μg/mL
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5.4.3 Oligonucleotide Concentration Expressed in pmol/μL
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5.5 Measuring RNA Concentration
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5.6 Molecular Weight, Molarity, and Nucleic Acid Length
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5.7 Estimating DNA Concentration on an Ethidium Bromide-Stained Gel
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Chapter Summary
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CHAPTER 6 Labeling Nucleic Acids with Radioisotopes
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Introduction
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6.1 Units of Radioactivity – The Curie (Ci)
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6.2 Estimating Plasmid Copy Number
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6.3 Labeling DNA by Nick Translation
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6.3.1 Determining Percent Incorporation of Radioactive Label from Nick Translation
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6.3.2 Calculating Specific Radioactivity of a Nick Translation Product
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6.4 Random Primer Labeling of DNA
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6.4.1 Random Primer Labeling – Percent Incorporation
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6.4.2 Random Primer Labeling – Calculating Theoretical Yield
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6.4.3 Random Primer Labeling – Calculating Actual Yield
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6.4.4 Random Primer Labeling – Calculating Specific Activity of the Product
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6.5 Labeling 3′ Termini with Terminal Transferase
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6.5.1 3′-end Labeling with Terminal Transferase – Percent Incorporation
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6.5.2 3′-end Labeling with Terminal Transferase – Specific Activity of the Product
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6.6 Complementary DNA (cDNA) Synthesis
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6.6.1 First Strand cDNA Synthesis
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6.6.2 Second Strand cDNA Synthesis
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6.7 Homopolymeric Tailing
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6.8 In Vitro Transcription
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Chapter Summary
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CHAPTER 7 Oligonucleotide Synthesis
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Introduction
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7.1 Synthesis Yield
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7.2 Measuring Stepwise and Overall Yield by the Dimethoxytrityl (DMT) Cation Assay
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7.2.1 Overall Yield
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7.2.2 Stepwise Yield
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7.3 Calculating Micromoles of Nucleoside Added at Each Base Addition Step
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Chapter Summary
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CHAPTER 8 The Polymerase Chain Reaction (PCR)
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Introduction
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8.1 Template and Amplification
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8.2 Exponential Amplification
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8.3 Polymerase Chain Reaction (PCR) Efficiency
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8.4 Calculating the T[sub(m)] of the Target Sequence
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8.5 Primers
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8.6 Primer T[sub(m)]
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8.6.1 Calculating T[sub(m)] Based on Salt Concentration, G/C Content, and DNA Length
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8.6.2 Calculating T[sub(m)] Based on Nearest-Neighbor Interactions
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8.7 Deoxynucleoside Triphosphates (dNTPs)
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8.8 DNA Polymerase
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8.8.1 Calculating DNA Polymerase's Error Rate
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8.9 Quantitative Polymerase Chain Reaction (PCR)
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Chapter Summary
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References
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Further Reading
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CHAPTER 9 The Real-time Polymerase Chain Reaction (RT-PCR)
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Introduction
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9.1 The Phases of Real-time PCR
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9.2 Controls
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9.3 Absolute Quantification by the TaqMan Assay
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9.3.1 Preparing the Standards
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9.3.2 Preparing a Standard Curve for Quantitative Polymerase Chain Reaction (qPCR) Based on Gene Copy Number
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9.3.3 The Standard Curve
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9.3.4 Standard Deviation
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9.3.5 Linear Regression and the Standard Curve
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9.4 Amplification Efficiency
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9.5 Measuring Gene Expression
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9.6 Relative Quantification – The ΔΔC[sub(T)] Method
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9.6.1 The 2[equation omitted] Method – Deciding on an Endogenous Reference
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9.6.2 The 2[equation omitted] Method – Amplification Efficiency
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9.6.3 The 2[equation omitted] Method – is the Reference Gene Affected by the Experimental Treatment?
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9.7 The Relative Standard Curve Method
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9.7.1 Standard Curve Method for Relative Quantitation
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9.8 Relative Quantification by Reaction Kinetics
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9.9 The R[sub(0)] Method of Relative Quantification
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9.10 The Pfaffl Model
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Chapter Summary
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References
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Further Reading
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CHAPTER 10 Recombinant DNA
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Introduction
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10.1 Restriction Endonucleases
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10.1.1 The Frequency of Restriction Endonuclease Cut Sites
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10.2 Calculating the Amount of Fragment Ends
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10.2.3 The Amount of Ends Generated by Multiple Cuts
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10.3 Ligation
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10.3.1 Ligation Using λ-Derived Vectors
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10.3.2 Packaging of Recombinant λ Genomes
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10.3.3 Ligation Using Plasmid Vectors
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10.3.4 Transformation Efficiency
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10.4 Genomic Libraries – How Many Clones Do You Need?
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10.5 cDNA Libraries – How Many Clones are Enough?
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10.6 Expression Libraries
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10.7 Screening Recombinant Libraries by Hybridization to DNA Probes
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10.7.1 Oligonucleotide Probes
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10.7.2 Hybridization Conditions
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10.7.3 Hybridization Using Double-Stranded DNA (dsDNA) Probes
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10.8 Sizing DNA Fragments by Gel Electrophoresis
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10.9 Generating Nested Deletions Using Nuclease BAL 31
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Chapter Summary
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References
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CHAPTER 11 Protein
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Introduction
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11.1 Calculating a Protein's Molecular Weight from Its Sequence
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11.2 Protein Quantication by Measuring Absorbance at 280 nm
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11.3 Using Absorbance Coefficients and Extinction Coefficients to Estimate Protein Concentration
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11.3.1 Relating Absorbance Coefficient to Molar Extinction Coefficient
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11.3.2 Determining a Protein's Extinction Coefficient
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11.4 Relating Concentration in Milligrams Per Milliliter to Molarity
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11.5 Protein Quantitation Using A[sub(280)] When Contaminating Nucleic Acids are Present
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11.6 Protein Quantification at 205 nm
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11.7 Protein Quantitation at 205 nm When Contaminating Nucleic Acids are Present
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11.8 Measuring Protein Concentration by Colorimetric Assay – The Bradford Assay
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11.9 Using β-Galactosidase to Monitor Promoter Activity and Gene Expression
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11.9.1 Assaying β-Galactosidase in Cell Culture
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11.9.2 Specific Activity
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11.9.3 Assaying β-Galactosidase from Purified Cell Extracts
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11.10 Thin Layer Chromatography (TLC) and the Retention Factor (R[sub(f)])
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11.11 Estimating a Protein's Molecular Weight by Gel Filtration
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11.12 The Chloramphenicol Acetyltransferase (CAT) Assay
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11.12.1 Calculating Molecules of Chloramphenicol Acetyltransferase (CAT)
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11.13 Use of Luciferase in a Reporter Assay
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11.14 In Vitro Translation – Determining Amino Acid Incorporation
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11.15 The Isoelectric Point (pI) of a Protein
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Chapter Summary
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References
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Further Reading
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CHAPTER 12 Centrifugation
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Introduction
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12.1 Relative Centrifugal Force (RCF) (g Force)
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12.1.1 Converting g Force to Revolutions Per Minute (rpm)
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12.1.2 Determining g Force and Revolutions Per Minute (rpm) by Use of a Nomogram
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12.2 Calculating Sedimentation Times
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Chapter Summary
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References
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Further Reading
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CHAPTER 13 Forensics and Paternity
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Introduction
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13.1 Alleles and Genotypes
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13.1.1 Calculating Genotype Frequencies
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13.1.2 Calculating Allele Frequencies
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13.2 The Hardy–Weinberg Equation and Calculating Expected Genotype Frequencies
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13.3 The Chi-Square Test – Comparing Observed to Expected Values
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13.3.1 Sample Variance
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13.3.2 Sample Standard Deviation
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13.4 The Power of Inclusion (P[sub(i)])
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13.5 The Power of Discrimination (P[sub(d)])
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13.6 DNA Typing and Weighted Average
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13.7 The Multiplication Rule
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13.8 The Paternity Index (PI)
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13.8.1 Calculating the Paternity Index (PI) When the Mother's Genotype is not Available
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13.8.2 The Combined Paternity Index (CPI)
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Chapter Summary
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References
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Further Reading
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Appendix A
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Index
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A
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B
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C
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D
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E
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F
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G
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H
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I
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L
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M
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N
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O
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P
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Q
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R
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S
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T
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V
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Z
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