 |
Current protocols essential laboratory techniques / edited by Sean R. Gallagher, Emily A. Wiley. — 2nd ed. — Hoboken : John Wiley & Sons, c2012. – (61.057/C976/2nd ed.) |
Contents
Contents
Foreword
Preface
About the Second Edition and Online Access
How to Use This Manual
Acknowledgments
Access to Current Protocols Essential Laboratory Techniques Online
Contributors
Common Conversion Factors
Introduction
Converting Units of Volume
Converting Temperatures
A Note About Writing Units of Measure
Internet Resources
Combining Techniques to Answer Molecular Questions
Introduction
Nucleic Acids
Proteins
Whole Cells and Subcellular Structures
General
Internet Resources
1 Volume/Weight Measurement
1.1 Volume Measurement
Overview and Principles
Micropipettors
Pipets
Volumetric Containers
Burets and Graduated Cylinders
Cleaning Volumetric Apparatus
Literature Cited
Internet Resources
1.2 Weight Measurement
Overview and Principles
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1: Measuring Mass Using a Top-Loading Balance
Basic Protocol 2: Measuring Mass Using an Analytical Balance
Understanding Results
Troubleshooting
Literature Cited
Internet Resources
2 Concentration Measurement
2.1 Spectrophotometry
Overview and Principles
Components of a Spectrophotometer
How a Spectrophotometer Works
Strategic Questions
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1 : Preparation of a Standard Curve for Dipicolinic Acid (DPA)
Basic Protocol 2: Determination of a Standard Curve for Dipicolinic Acid (DPA) and Measurement of DPA Concentration in Unknown Samples
Support Protocol: Optimizing Spectrophotometer Settings
Understanding Results
Troubleshooting
Literature Cited
Internet Resources
2.2 Quantitation of Nucleic Acids and Proteins
Overview and Principles
Strategic Questions
Strategic Planning
Protocols: Nucleic Acid Quantification
Basic Protocol 3: Traditional Detection of Nucleic Acids Using Absorption Spectroscopy
Basic Protocol 2: Microvolume Detection of Nucleic Acids Using Absorption Spectroscopy
Alternate Protocol 1 : DNA Detection Using the DNA-Binding Fluorochrome Hoechst 33258
Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence
Alternate Protocol 3: DNA Detection Using PicoGreen dsDNA Quantitation Reagent
Protocols: Protein Quantification
Basic Protocol 3: Lowry Protein Assay
Alternate Protocol 4: Lowry Protein Assay， Reduced Volume
Support Protocol: Deoxycholate-Trichloroacetic Acid (DOC-TCA) Sample Precipitation for Removal of Interfering Compounds and Sample Concentration
Basic Protocol 4: BCA Protein Assay
Basic Protocol 5: Coomassie Blue Protein Assay (Bradford Assay)
Basic Protocol 6: Traditional UV Spectrophotometry
Alternate Protocol 5: Protein Quantitation with UV Spectroscopy and Correction for Like-Acid Contamination
Basic Protocol 7: Microvolume UV Spectrophotometry
Protocol Common to Nucleic Acids and Proteins
Basic Protocol 8: Gel-Based Quantitation of Proteins and Nucleic Acids
Reagents and Solutions
Understanding Results
Troubleshooting
Variations
Literature Cited
Key References
Internet Resources
3 Reagent Preparation
3.1 Reagent Preparation: Theoretical and Practical Discussions
Reagent Preparation
Accuracy of Weighing and Pipetting
Use of Calibrated pH Meters
Avoiding Chemical and Microbial Contamination of Reagents
Preparing Reagent or Buffer Solutions
Making Buffer Solutions
Literature Cited
Internet Resources
3.2 Measurement of pH
Overview and Principles
Equipment for Measuring pH
pH Electrode Care
Measurement of pH
Troubleshooting
Internet Resources
3.3 Recipes for Commonly Encountered Reagents
Introduction
Recipes
Literature Cited
Internet Resources
4 Cell Culture Techniques
4.1 Aseptic Technique
Overview and Principles
Strategic Planning
Safety Considerations
Techniques for Maintaining Aseptic Conditions
Literature Cited
Internet Resources
4.2 Culture of Escherichia coli and Related Bacteria
Overview and Principles
Safety Considerations
Commonly Used Tools
Reagents
Culturing Bacteria on Solid Media
Growing Bacteria in Liquid Culture
Analysis and Purification of Plasmid DNAs
Storage of Cultures
Commonly Used Bacterial Media
Literature Cited
Internet Resources
5 Sample Preparation
5.1 Centrifugation
Introduction
Definitions
Rotors
Literature Cited
5.2 Purification and Concentration of Nucleic Acids
Overview and Principles
Strategic Questions
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1: Phenol Extraction and Ethanol Precipitation of DNA
Alternate Protocol 1: Purification of Plasmid DNA Using Silica Membrane Spin Columns
Support Protocol 1: Precipitation of DNA Using Isopropanol
Support Protocol 2: Concentration of DNA Using Butanol
Basic Protocol 2: Single-Step RNA Isolation from Cultured Cells or Tissues
Alternate Protocol 2: Purification and Concentration of RNA and Dilute Solutions of DNA
Reagents and Solutions
Understanding Results
Troubleshooting
Acknowledgments
Literature Cited
Internet Resources
6 Chromatography
6.1 Overview of Chromatography
Overview and Principles
Common Types of Chromatography for Purification of Proteins
Literature Cited
Internet Resources
7 Electrophoresis
7.1 Overview of Electrophoresis
Overview and Principles
Strategic Questions
General Concepts
Visualization of Resolved Molecules
Imaging Resolved Molecules
Literature Cited
Internet Resources
7.2 Agarose Gel Electrophoresis
Overview and Principles
Strategic Questions
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1: DNA Agarose Gel Electrophoresis
Basic Protocol 2: Denaturing RNA Agarose Gel Electrophoresis
Reagents and Solutions
Understanding Results
Troubleshooting
Variations
Acknowledgments
Literature Cited
Internet Resources
7.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Overview and Principles
Strategic Questions
Strategic Planning
Safety Considerations
Protocols
Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis: The Laemmli Gel Method
Support Protocol 1 : Casting a Gel for Use in Denaturing Discontinuous Electrophoresis
Support Protocol 2: Calculating Molecular Mass
Support Protocol 3: Recrystallizing SDS
Reagents and Solutions
Understanding Results
Troubleshooting
Variations
Literature Cited
Key Reference
7.4 Staining Proteins in Gels
Overview and Principles
Strategic Questions
Strategic Planning
Protocols
Basic Protocol 1 : Coomassie Blue Staining
Alternate Protocol 1: Rapid Coomassie Blue Staining
Basic Protocol 2: Nonammoniacal Silver Staining
Alternate Protocol 2: Rapid Silver Staining
Basic Protocol 3: Fluorescent Staining Using SYPRO Orange or Red
Support Protocol: Photography of SYPRO Orange or Red Fluorescently Stained Gels
Reagents and Solutions
Understanding Results
Troubleshooting
Variations
Literature Cited
Internet Resources
8 Blotting
8.1 Overview of Blotting
General Overview
General Considerations
Southern and Northern Blotting
Immunoblotting
Literature Cited
Internet Resources
8.2 Nucleic Acid Blotting: Southern and Northern
Overview and Principles
Strategic Questions
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1 : Southern Blotting
Basic Protocol 2: Northern Blotting
Support Protocol: Assembling a Blotting Transfer Apparatus
Reagents and Solutions
Understanding Results
Troubleshooting
Variations
Literature Cited
Key References
Internet Resources
8.3 Protein Blotting: Immunoblotting
Overview and Principles
Strategic Questions
Strategic Planning
Protocols
Basic Protocol 1 : Protein Blotting with Semidry Systems
Basic Protocol 2: lank Transfer
Alternate Protocol 1 : Slot and Dot Blotting
Support Protocol 1: Ponceau S Staining of Transferred Proteins
Support Protocol 2: India Ink Staining of Transferred Proteins
Support Protocol 3: Gold Staining of Transferred Proteins
Support Protocol 4: Alkali Enhancement of Protein Staining
Support Protocol 5: Fluorescent Protein Blot Staining of Transferred Proteins
Support Protocol 6: Viewing and Photographing SYPRO Ruby-Stained Protein Blots
Basic Protocol 3: Immunoprobing with Directly Conjugated Secondary Antibody
Alternate Protocol 2: Immunoprobing with Avidin-Biotin Coupling to Secondary Antibody
Basic Protocol 4: Visualization with Chromogenic Substrates
Alternate Protocol 3: Visualization with Luminescent Substrates
Alternate Protocol 4: Fluorescent Blot Preparation and Analysis
Reagents and Solutions
Understanding Results
Troubleshooting
Variations
Literature Cited
Internet Resources
8.4 Labeling DNA and Preparing Probes
Overview and Principles
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1: 5' End-Labeling of DNA with T4 Polynucleotide Kinase
Basic Protocol 2: Labeling DNA by Nick Translation
Basic Protocol 3: Labeling DNA by Random Primed Synthesis
Support Protocol: Purification of Labeled Probes Using Gel-Filtration Spin Columns
Reagents and Solutions
Understanding Results
Troubleshooting
Variations
Literature Cited
Internet Resources
9 Microscopy
9.1 Conventional Light Microscopy
Parts of the Light Microscope
Care and Maintenance
Basic Principles and Definitions
Magnification Versus Resolution
Getting Comfortable
Finding the Object to be Viewed
Marking the Location of an Object: Secrets of the Microscope Stage
A Quick Guide to Choosing from Various Optical Techniques
K6hler Illumination: Secrets of the Substage Condenser
Oil Immersion
Dark-Field， Rheinberg， Polarized-Light， Phase-Contrast， and DIC Microscopy
Acknowledgments
Literature Cited
Key References
Internet Resources
9.2 Immunofluorescence Microscopy
Overview and Principles
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1 : Processing Fibroblasts
Basic Protocol 2: Processing Tetrahymena Cells
Alternate Protocol: Staining Cells Adhered to Poly-L-Lysine-Coated Coverslips
Basic Protocol 3: Visualizing the Cells
Reagents and Solutions
Understanding Results
Troubleshooting
Acknowledgments
Literature Cited
Internet Resources
10 Enzymatic Reactions
10.1 Working with Enzymes
Introduction
Overview of Enzymes
Handling Enzymes in the Laboratory
Example of Setting Up an Enzymatic Reaction: Restriction Enzymes
Literature Cited
Key References
Internet Resources
10.2 Overview of PCR
Overview and Principles
Strategic Planning
Strategic Questions
Safety Considerations
Protocols
Basic Protocol: Routine PCR
Support Protocol 1 : Using Temperature Gradients for Rapid Optimization of PCR Cycling Conditions
Support Protocol 2: Titration of MgCI2 Concentration
Reagents and Solutions
Understanding Results
Troubleshooting
Variations
Literature Cited
Internet Resources
10.3 Real-Time PCR
Overview and Principles
Strategic Questions
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1: Synthesis of cDNA by Reverse Transcription
Basic Protocol 2: Real-Time PCR Amplification and Analysis
Support Protocol 1: Determination of Amplification Efficiency
Support Protocol 2: Analyzing Results Using the Pfaffl Method to Calculate Fold Induction
Support Protocol 3: Serial Dilution for Standard Curve
Understanding Results
Troubleshooting
Variations
Literature Cited
Internet Resources
11 Bioinformatics
11.1 Using NCBI BLAST
Overview and Principles
Strategic Questions
Strategic Planning
Protocols
Basic Protocol 1 : Search a Nucleotide Database Using a Nucleotide Query: Nucleotide BLAST (BLASTN)
Basic Protocol 2: Search a Protein Database Using a Protein Query: Protein BLAST (BLASTP)
Basic Protocol 3: Search a Protein Database Using a Translated Nucleotide Query: BLASTX
Basic Protocol 4: Searching a Translated Nucleotide Database Using a Protein or Nucleotide Query: TBLASTN and TBLASTX
Understanding Results
Troubleshooting
Literature Cited
Internet Resources
