Calculations for Molecular Biology and Biotechnology

 
 
Academic Press
  • 3. Auflage
  • |
  • erschienen am 16. Juni 2016
  • |
  • 496 Seiten
 
E-Book | ePUB mit Adobe DRM | Systemvoraussetzungen
E-Book | PDF mit Adobe DRM | Systemvoraussetzungen
978-0-12-802598-7 (ISBN)
 

Calculations in Molecular Biology and Biotechnology, Third Edition, helps researchers utilizing molecular biology and biotechnology techniques-from student to professional-understand which type of calculation to use and why. Research in biotechnology and molecular biology requires a vast amount of calculations. Results of one data set become the basis of the next. An error of choosing the wrong type of equation can turn what would have been a successful research project or weeks of labor and research into a veritable house of cards. It could be how you calculated the medium in which you test your sample to calculating how long it takes a sample to grow to calculating the synthesis of multiple variables.

In one easy to use reference, Stephenson reviews the mathematics and statistics related to the day-to-day functions of biotechnology and molecular biology labs, which is a sticking point for many students, technicians, and researchers. The book covers all of the basic mathematical and statistical needs for students and professionals, providing them with a useful tool for their work.


  • Features comprehensive calculations in biotechnology and molecular biology experiments from start to finish
  • Provides coverage ranging from basic scientific notations to complex subjects like nucleic acid chemistry and recombinant DNA technology
  • Includes recent applications of the procedures and computations in clinical, academic, industrial, and basic research laboratories cited throughout the text
  • Features new coverage of digital PCR and protein quantification including chromatography and radiolabelling of proteins
  • Includes more sample problems in every chapter for readers to practice concepts


Frank Stephenson received his doctorate in molecular biology from UC Berkeley and has published several books in the field including 'DNA: How the Biotech Revolution is Changing the Way We Fight Disease' and 'A Hands-On Introduction to Forensic Science: Cracking the Case'. He is currently an instructor in the Technical Training Department with ThermoFisher Scientific, the world's leading manufacturer of instrumentation and reagents for the biotechnology industry.
  • Englisch
  • Saint Louis
  • |
  • USA
Elsevier Science
  • 8,02 MB
978-0-12-802598-7 (9780128025987)
0128025980 (0128025980)
weitere Ausgaben werden ermittelt
  • Front Cover
  • Calculations for Molecular Biology and Biotechnology
  • Calculations for Molecular Biology and Biotechnology
  • Copyright
  • Contents
  • Biography
  • 1 - Scientific Notation and Metric Prefixes
  • INTRODUCTION
  • 1.1 SIGNIFICANT DIGITS
  • 1.1.1 Rounding Off Significant Digits in Calculations
  • 1.2 EXPONENTS AND SCIENTIFIC NOTATION
  • 1.2.1 Expressing Numbers in Scientific Notation
  • 1.2.2 Converting Numbers From Scientific Notation to Decimal Notation
  • 1.2.3 Adding and Subtracting Numbers Written in Scientific Notation
  • 1.2.4 Multiplying and Dividing Numbers Written in Scientific Notation
  • 1.3 METRIC PREFIXES
  • 1.3.1 Conversion Factors and Canceling Terms
  • CHAPTER SUMMARY
  • 2 - Solutions, Mixtures, and Media
  • INTRODUCTION
  • 2.1 CALCULATING DILUTIONS: A GENERAL APPROACH
  • 2.2 CONCENTRATIONS BY A FACTOR OF X
  • 2.3 PREPARING PERCENT SOLUTIONS
  • 2.4 DILUTING PERCENT SOLUTIONS
  • 2.5 MOLES AND MOLECULAR WEIGHT-DEFINITIONS
  • 2.5.1 Molarity
  • 2.5.2 Preparing Molar Solutions in Water With Hydrated Compounds
  • 2.5.3 Diluting Molar Solutions
  • 2.5.4 Converting Molarity to Percent
  • 2.5.5 Converting Percent to Molarity
  • 2.6 NORMALITY
  • 2.7 PH
  • 2.8 PKA AND THE HENDERSON-HASSELBALCH EQUATION
  • CHAPTER SUMMARY
  • 3 - Cell Growth
  • 3.1 THE BACTERIAL GROWTH CURVE
  • 3.1.1 Sample Data
  • 3.2 MANIPULATING CELL CONCENTRATION
  • 3.3 PLOTTING OD550 VERSUS TIME ON A LINEAR GRAPH
  • 3.4 PLOTTING THE LOGARITHM OF OD550 VERSUS TIME ON A LINEAR GRAPH
  • 3.4.1 Logarithms
  • 3.4.2 Sample OD550 Data Converted to Log Values
  • 3.4.3 Plotting Log OD550 Versus Time
  • 3.5 PLOTTING THE LOG OF CELL CONCENTRATION VERSUS TIME
  • 3.5.1 Determining Log Values
  • 3.6 CALCULATING GENERATION TIME
  • 3.6.1 Slope and the Growth Constant
  • 3.6.2 Generation Time
  • 3.7 PLOTTING CELL GROWTH DATA ON A SEMILOG GRAPH
  • 3.7.1 Plotting OD550 Versus Time on a Semilog Graph
  • 3.7.2 Estimating Generation Time From a Semilog Plot of OD550 Versus Time
  • 3.8 PLOTTING CELL CONCENTRATION VERSUS TIME ON A SEMILOG GRAPH
  • 3.9 DETERMINING GENERATION TIME DIRECTLY FROM A SEMILOG PLOT OF CELL CONCENTRATION VERSUS TIME
  • 3.10 PLOTTING CELL DENSITY VERSUS OD550 ON A SEMILOG GRAPH
  • 3.11 THE FLUCTUATION TEST
  • 3.11.1 Fluctuation Test Example
  • 3.11.1.1 Interpretation of Results
  • 3.11.2 Variance
  • 3.12 MEASURING MUTATION RATE
  • 3.12.1 The Poisson Distribution
  • 3.12.2 Calculating Mutation Rate by Using the Poisson Distribution
  • 3.12.3 Using a Graphical Approach to Calculate Mutation Rate From Fluctuation Test Data
  • 3.12.4 Mutation Rate Determined by Plate Spreading
  • 3.13 MEASURING CELL CONCENTRATION ON A HEMOCYTOMETER
  • CHAPTER SUMMARY
  • REFERENCE
  • 4 - Working with Bacteriophage
  • INTRODUCTION
  • 4.1 MULTIPLICITY OF INFECTION
  • 4.2 PROBABILITIES AND MULTIPLICITY OF INFECTION
  • 4.3 MEASURING PHAGE TITER
  • 4.4 DILUTING BACTERIOPHAGE
  • 4.5 MEASURING BURST SIZE
  • CHAPTER SUMMARY
  • 5 - Nucleic Acid Quantification
  • 5.1 QUANTIFICATION OF NUCLEIC ACIDS BY ULTRAVIOLET SPECTROSCOPY
  • 5.2 DETERMINING THE CONCENTRATION OF DOUBLE-STRANDED DNA
  • 5.2.1 Using Absorbance and an Extinction Coefficient to Calculate Double-Stranded DNA Concentration
  • 5.2.2 Calculating DNA Concentration as a Millimolar Amount
  • 5.2.3 Using PicoGreen® to Determine DNA Concentration
  • 5.3 DETERMINING THE CONCENTRATION OF SINGLE-STRANDED DNA MOLECULES
  • 5.3.1 Single-Stranded DNA Concentration Expressed in µg/mL
  • 5.3.2 Determining the Concentration of High-Molecular-Weight Single-Stranded DNA in Picomoles per Microliter
  • 5.3.3 Expressing ssDNA Concentration as a Millimolarity Amount
  • 5.4 OLIGONUCLEOTIDE QUANTIFICATION
  • 5.4.1 Optical Density Units
  • 5.4.2 Expressing an Oligonucleotide's Concentration in µg/mL
  • 5.4.3 Oligonucleotide Concentration Expressed in Picomoles per Microliter
  • 5.5 MEASURING RNA CONCENTRATION
  • 5.6 MOLECULAR WEIGHT, MOLARITY, AND NUCLEIC ACID LENGTH
  • 5.7 ESTIMATING DNA CONCENTRATION ON AN ETHIDIUM BROMIDE-STAINED GEL
  • 5.8 DYE-LABELED NUCLEIC ACIDS
  • 5.9 LIMIT OF DETECTION AND LIMIT OF QUANTITATION
  • CHAPTER SUMMARY
  • 6 - Labeling Nucleic Acids With Radioisotopes
  • INTRODUCTION
  • 6.1 UNITS OF RADIOACTIVITY: THE CURIE
  • 6.2 ESTIMATING PLASMID COPY NUMBER
  • 6.3 LABELING DNA BY NICK TRANSLATION
  • 6.3.1 Determining Percent Incorporation of Radioactive Label From Nick Translation
  • 6.3.2 Calculating Specific Radioactivity of a Nick Translation Product
  • 6.4 RANDOM PRIMER LABELING OF DNA
  • 6.4.1 Random Primer Labeling-Percent Incorporation
  • 6.4.2 Random Primer Labeling-Calculating Theoretical Yield
  • 6.4.3 Random Primer Labeling-Calculating Actual Yield
  • 6.4.4 Random Primer Labeling-Calculating Specific Activity of the Product
  • 6.5 LABELING 3´ TERMINI WITH TERMINAL TRANSFERASE
  • 6.5.1 3´-End Labeling With Terminal Transferase-Percent Incorporation
  • 6.5.2 3´-End Labeling With Terminal Transferase-Specific Activity of the Product
  • 6.6 COMPLEMENTARY DNA SYNTHESIS
  • 6.6.1 First Strand Complementary DNA Synthesis
  • 6.6.2 Second Strand Complementary DNA Synthesis
  • 6.7 HOMOPOLYMERIC TAILING
  • 6.8 IN VITRO TRANSCRIPTION
  • CHAPTER SUMMARY
  • 7 - Oligonucleotide Synthesis
  • INTRODUCTION
  • 7.1 SYNTHESIS YIELD
  • 7.2 MEASURING STEPWISE AND OVERALL YIELD BY THE DIMETHOXYTRITYL CATION ASSAY
  • 7.2.1 Overall Yield
  • 7.2.2 Stepwise Yield
  • 7.3 CALCULATING MICROMOLES OF NUCLEOSIDE ADDED AT EACH BASE ADDITION STEP
  • CHAPTER SUMMARY
  • 8 - The Polymerase Chain Reaction
  • INTRODUCTION
  • 8.1 TEMPLATE AND AMPLIFICATION
  • 8.2 EXPONENTIAL AMPLIFICATION
  • 8.3 POLYMERASE CHAIN REACTION EFFICIENCY
  • 8.4 CALCULATING THE TM OF THE TARGET SEQUENCE
  • 8.5 PRIMERS
  • 8.6 PRIMER TM
  • 8.6.1 Calculating Tm Based on Salt Concentration, G/C Content, and DNA Length
  • 8.6.2 Calculating Tm Based on Nearest-Neighbor Interactions
  • 8.7 DEOXYNUCLEOSIDE TRIPHOSPHATES
  • 8.8 DNA POLYMERASE
  • 8.8.1 Calculating DNA Polymerase's Error Rate
  • 8.9 QUANTITATIVE PCR
  • CHAPTER SUMMARY
  • REFERENCES
  • FURTHER READING
  • 9 - Real-Time PCR
  • INTRODUCTION
  • 9.1 THE PHASES OF A REAL-TIME POLYMERASE CHAIN REACTION
  • 9.2 CONTROLS
  • 9.3 ABSOLUTE QUANTIFICATION BY THE TAQMAN ASSAY
  • 9.3.1 Preparing the Standards
  • 9.3.2 Preparing a Standard Curve for Quantitative Polymerase Chain Reaction Based on Gene Copy Number
  • 9.3.3 The Standard Curve
  • 9.3.4 Standard Deviation
  • 9.3.5 Linear Regression and the Standard Curve
  • 9.4 AMPLIFICATION EFFICIENCY
  • 9.5 MEASURING GENE EXPRESSION
  • 9.6 RELATIVE QUANTIFICATION: THE ??CT METHOD
  • 9.6.1 The 2-??CT Method-Deciding on an Endogenous Reference
  • 9.6.2 The 2-??CT Method-Amplification Efficiency
  • 9.6.3 The 2-??CT Method: Is the Reference Gene Affected by the Experimental Treatment?
  • 9.7 RELATIVE STANDARD CURVE METHOD
  • 9.7.1 Standard Curve Method for Relative Quantitation
  • 9.8 RELATIVE QUANTIFICATION BY REACTION KINETICS
  • 9.9 THE R0 METHOD OF RELATIVE QUANTIFICATION
  • 9.10 THE PFAFFL MODEL
  • 9.11 DIGITAL POLYMERASE CHAIN REACTION
  • 9.11.1 Confidence Interval
  • CHAPTER SUMMARY
  • REFERENCES
  • FURTHER READING
  • 10 - Recombinant DNA
  • INTRODUCTION
  • 10.1 RESTRICTION ENDONUCLEASES
  • 10.1.1 The Frequency of Restriction Endonuclease Cut Sites
  • 10.2 CALCULATING THE AMOUNT OF FRAGMENT ENDS
  • 10.2.1 The Amount of Ends Generated by Multiple Cuts
  • 10.3 LIGATION
  • 10.3.1 Ligation Using ?-Derived Vectors
  • 10.3.2 Packaging of Recombinant ? Genomes
  • 10.3.3 Ligation Using Plasmid Vectors
  • 10.3.4 Transformation Efficiency
  • 10.4 GENOMIC LIBRARIES-HOW MANY CLONES DO YOU NEED?
  • 10.5 CDNA LIBRARIES-HOW MANY CLONES ARE ENOUGH?
  • 10.6 EXPRESSION LIBRARIES
  • 10.7 SCREENING RECOMBINANT LIBRARIES BY HYBRIDIZATION TO DNA PROBES
  • 10.7.1 Oligonucleotide Probes
  • 10.7.2 Hybridization Conditions
  • 10.7.3 Hybridization Using Double-Stranded DNA Probes
  • 10.8 SIZING DNA FRAGMENTS BY GEL ELECTROPHORESIS
  • 10.9 GENERATING NESTED DELETIONS USING NUCLEASE BAL 31
  • CHAPTER SUMMARY
  • REFERENCES
  • 11 - Protein
  • INTRODUCTION
  • 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
  • 11.3 PROTEIN QUANTIFICATION BY MEASURING ABSORBANCE AT 280NM
  • 11.4 USING ABSORBANCE COEFFICIENTS AND EXTINCTION COEFFICIENTS TO ESTIMATE PROTEIN CONCENTRATION
  • 11.4.1 Relating Absorbance Coefficient to Molar Extinction Coefficient
  • 11.4.2 Determining a Protein's Extinction Coefficient
  • 11.5 RELATING CONCENTRATION IN MILLIGRAMS PER MILLILITER TO MOLARITY
  • 11.6 PROTEIN QUANTITATION USING A280 WHEN CONTAMINATING NUCLEIC ACIDS ARE PRESENT
  • 11.7 PROTEIN QUANTIFICATION AT 205NM
  • 11.8 PROTEIN QUANTITATION AT 205NM WHEN CONTAMINATING NUCLEIC ACIDS ARE PRESENT
  • 11.9 LABELING PROTEINS WITH FLUORESCENT DYES
  • 11.10 MEASURING PROTEIN CONCENTRATION BY COLORIMETRIC ASSAY-THE BRADFORD ASSAY
  • 11.11 THIN-LAYER CHROMATOGRAPHY AND RF
  • 11.12 ESTIMATING A PROTEIN'S MOLECULAR WEIGHT BY GEL FILTRATION
  • 11.13 PROTEIN VOLUME
  • 11.14 USING ß-GALACTOSIDASE TO MONITOR PROMOTER ACTIVITY AND GENE EXPRESSION
  • 11.14.1 Assaying ß-Galactosidase in Cell Culture
  • 11.14.2 Specific Activity
  • 11.14.3 Assaying ß-Galactosidase From Purified Cell Extracts
  • 11.15 THE CHLORAMPHENICOL ACETYLTRANSFERASE ASSAY
  • 11.15.1 Calculating Molecules of Chloramphenicol Acetyltransferase
  • 11.16 USE OF LUCIFERASE IN A REPORTER ASSAY
  • 11.17 DNA POLYMERASE ACTIVITY
  • 11.18 IN VITRO TRANSLATION-DETERMINING AMINO ACID INCORPORATION
  • 11.19 THE ISOELECTRIC POINT OF A PROTEIN
  • CHAPTER SUMMARY
  • REFERENCES
  • FURTHER READING
  • 12 - Centrifugation
  • INTRODUCTION
  • 12.1 RELATIVE CENTRIFUGAL FORCE (G FORCE)
  • 12.1.1 Converting g Force to Revolutions per Minute
  • 12.1.2 Determining g Force and Revolutions per Minute by Use of a Nomogram
  • 12.2 CALCULATING SEDIMENTATION TIMES
  • CHAPTER SUMMARY
  • FURTHER READING
  • 13 - Forensics and Paternity
  • INTRODUCTION
  • 13.1 ALLELES AND GENOTYPES
  • 13.1.1 Calculating Genotype Frequencies
  • 13.1.2 Calculating Allele Frequencies
  • 13.2 THE HARDY-WEINBERG EQUATION AND CALCULATING EXPECTED GENOTYPE FREQUENCIES
  • 13.3 THE CHI-SQUARE TEST-COMPARING OBSERVED TO EXPECTED VALUES
  • 13.3.1 Sample Variance
  • 13.3.2 Sample Standard Deviation
  • 13.4 THE POWER OF INCLUSION
  • 13.5 THE POWER OF DISCRIMINATION
  • 13.6 DNA TYPING AND A WEIGHTED AVERAGE
  • 13.7 THE MULTIPLICATION RULE
  • 13.8 THE PATERNITY INDEX
  • 13.8.1 Calculating the Paternity Index When the Mother's Genotype Is Not Available
  • 13.8.2 The Combined Paternity Index
  • CHAPTER SUMMARY
  • MORE PROBLEMS
  • REFERENCES
  • FURTHER READING
  • A - Using Microsoft Excel's Graphing Utility
  • Index
  • A
  • B
  • C
  • D
  • E
  • F
  • G
  • H
  • I
  • L
  • M
  • N
  • O
  • P
  • Q
  • R
  • S
  • T
  • V
  • Z
  • Back Cover

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