| Foreword |
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xi | |
| Preface |
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xiii | |
| About the Second Edition and Online Access |
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xiii | |
| How to Use This Manual |
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xiv | |
| Acknowledgments |
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xvi | |
| Access to Current Protocols Essential Laboratory Techniques Online |
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xvii | |
| Contributors |
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xxi | |
| Common Conversion Factors |
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xxiii | |
| Introduction |
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xxiii | |
| Converting Units of Volume |
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xxiii | |
| Converting Temperatures |
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xxiv | |
| A Note About Writing Units of Measure |
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xxvii | |
| Internet Resources |
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xxviii | |
| Combining Techniques to Answer Molecular Questions |
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xxix | |
| Introduction |
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xxix | |
| Nucleic Acids |
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xxix | |
| Proteins |
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xxxi | |
| Whole Cells and Subcellular Structures |
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xxxii | |
| General |
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xxxii | |
| Internet Resources |
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xxxiii | |
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1 Volume/Weight Measurement |
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1 | (4) |
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5 | (3) |
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8 | (3) |
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11 | (1) |
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Burets and Graduated Cylinders |
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12 | (1) |
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Cleaning Volumetric Apparatus |
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12 | (2) |
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14 | (1) |
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14 | |
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1 | (2) |
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3 | (6) |
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9 | (1) |
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10 | (1) |
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Basic Protocol 1 Measuring Mass Using a Top-Loading Balance |
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10 | (1) |
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Basic Protocol 2 Measuring Mass Using an Analytical Balance |
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10 | (1) |
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10 | (1) |
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11 | (1) |
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11 | (1) |
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11 | |
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2 Concentration Measurement |
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1 | (3) |
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Components of a Spectrophotometer |
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4 | (4) |
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How a Spectrophotometer Works |
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8 | (5) |
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13 | (2) |
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15 | (4) |
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19 | (1) |
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19 | (1) |
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Basic Protocol 1 Preparation of a Standard Curve for Dipicolinic Acid (DPA) |
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19 | (2) |
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Basic Protocol 2 Determination of a Standard Curve for Dipicolinic Acid (DPA) and Measurement of DPA Concentration in Unknown Samples |
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21 | (5) |
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Support Protocol: Optimizing Spectrophotometer Settings |
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26 | (1) |
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27 | (1) |
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27 | (1) |
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28 | (1) |
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28 | |
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2.2 Quantitation of Nucleic Acids and Proteins |
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1 | (6) |
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7 | (1) |
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8 | (5) |
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Protocols: Nucleic Acid Quantification |
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13 | (1) |
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Basic Protocol 1 Traditional Detection of Nucleic Acids Using Absorption Spectroscopy |
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13 | (3) |
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Basic Protocol 2 Microvolume Detection of Nucleic Acids Using Absorption Spectroscopy |
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16 | (3) |
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Alternate Protocol 1 DNA Detection Using the DNA-Binding Fluorochrome Hoechst 33258 |
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19 | (1) |
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Alternate Protocol 2 DNA and RNA Detection with Ethidium Bromide Fluorescence |
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20 | (1) |
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Alternate Protocol 3 DNA Detection Using PicoGreen dsDNA Quantitation Reagent |
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21 | (4) |
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Protocols: Protein Quantification |
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25 | (1) |
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Basic Protocol 3 Lowry Protein Assay |
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25 | (1) |
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Alternate Protocol 4 Lowry Protein Assay, Reduced Volume |
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26 | (1) |
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Support Protocol: Deoxycholate-Trichloroacetic Acid (DOC-TCA) Sample Precipitation for Removal of Interfering Compounds and Sample Concentration |
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27 | (1) |
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Basic Protocol 4 BCA Protein Assay |
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27 | (1) |
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Basic Protocol 5 Coomassie Blue Protein Assay (Bradford Assay) |
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28 | (1) |
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Basic Protocol 6 Traditional UV Spectrophotometry |
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29 | (1) |
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Alternate Protocol 5 Protein Quantitation with UV Spectroscopy and Correction for Like-Acid Contamination |
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29 | (1) |
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Basic Protocol 7 Microvolume UV Spectrophotometry |
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30 | (2) |
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Protocol Common to Nucleic Acids and Proteins |
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32 | (1) |
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Basic Protocol 8 Gel-Based Quantitation of Proteins and Nucleic Acids |
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32 | (2) |
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34 | (2) |
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36 | (1) |
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37 | (1) |
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38 | (1) |
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38 | (1) |
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39 | (1) |
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39 | |
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3.1 Reagent Preparation: Theoretical and Practical Discussions |
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1 | (5) |
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Accuracy of Weighing and Pipetting |
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6 | (1) |
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Use of Calibrated pH Meters |
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6 | (1) |
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Avoiding Chemical and Microbial Contamination of Reagents |
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6 | (1) |
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Preparing Reagent or Buffer Solutions |
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7 | (4) |
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11 | (3) |
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14 | (1) |
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14 | |
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1 | (1) |
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Equipment for Measuring pH |
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2 | (7) |
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9 | (2) |
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11 | (3) |
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14 | (1) |
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15 | |
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3.3 Recipes for Commonly Encountered Reagents |
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1 | (1) |
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1 | (11) |
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12 | (1) |
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12 | |
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4 Cell Culture Techniques |
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1 | (1) |
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2 | (4) |
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6 | (1) |
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Techniques for Maintaining Aseptic Conditions |
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7 | (5) |
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12 | (1) |
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12 | |
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4.2 Culture of Escherichia coli and Related Bacteria |
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1 | (1) |
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2 | (1) |
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2 | (1) |
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3 | (1) |
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Culturing Bacteria on Solid Media |
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3 | (3) |
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Growing Bacteria in Liquid Culture |
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6 | (5) |
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Analysis and Purification of Plasmid DNAs |
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11 | (11) |
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22 | (1) |
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Commonly Used Bacterial Media |
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23 | (4) |
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27 | (1) |
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27 | |
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1 | (1) |
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1 | (4) |
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5 | (3) |
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8 | |
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5.2 Purification and Concentration of Nucleic Acids |
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1 | (5) |
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6 | (1) |
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7 | (1) |
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8 | (1) |
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8 | (1) |
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Basic Protocol 1 Phenol Extraction and Ethanol Precipitation of DNA |
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8 | (2) |
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Alternate Protocol 1 Purification of Plasmid DNA Using Silica Membrane Spin Columns |
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10 | (2) |
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Support Protocol 1 Precipitation of DNA Using Isopropanol |
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12 | (1) |
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Support Protocol 2 Concentration of DNA Using Butanol |
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13 | (1) |
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Basic Protocol 2 Single-Step RNA Isolation from Cultured Cells or Tissues |
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13 | (2) |
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Alternate Protocol 2 Purification and Concentration of RNA and Dilute Solutions of DNA |
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15 | (1) |
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16 | (1) |
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17 | (2) |
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19 | (2) |
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21 | (1) |
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21 | (1) |
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22 | |
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6.1 Overview of Chromatography |
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1 | (2) |
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Common Types of Chromatography for Purification of Proteins |
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3 | (7) |
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10 | (1) |
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10 | |
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7.1 Overview of Electrophoresis |
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1 | (1) |
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1 | (2) |
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3 | (2) |
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Visualization of Resolved Molecules |
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5 | (1) |
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Imaging Resolved Molecules |
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6 | (1) |
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7 | (1) |
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7 | |
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7.2 Agarose Gel Electrophoresis |
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1 | (2) |
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3 | (1) |
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3 | (2) |
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5 | (1) |
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6 | (1) |
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Basic Protocol 1 DNA Agarose Gel Electrophoresis |
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6 | (6) |
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Basic Protocol 2 Denaturing RNA Agarose Gel Electrophoresis |
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12 | (3) |
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15 | (1) |
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16 | (3) |
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19 | (1) |
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19 | (3) |
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22 | (1) |
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22 | (1) |
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22 | |
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7.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) |
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1 | (6) |
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7 | (1) |
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8 | (2) |
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10 | (1) |
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11 | (1) |
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Basic Protocol: Denaturing (SDS) Discontinuous Gel Electrophoresis: The Laemmli Gel Method |
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11 | (4) |
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Support Protocol 1 Casting a Gel for Use in Denaturing Discontinuous Electrophoresis |
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15 | (3) |
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Support Protocol 2 Calculating Molecular Mass |
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18 | (2) |
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Support Protocol 3 Recrystallizing SDS |
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20 | (1) |
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21 | (1) |
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22 | (1) |
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22 | (4) |
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26 | (2) |
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28 | (1) |
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28 | |
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7.4 Staining Proteins in Gels |
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1 | (1) |
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2 | (1) |
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3 | (1) |
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4 | (1) |
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Basic Protocol 1 Coomassie Blue Staining |
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4 | (1) |
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Alternate Protocol 1 Rapid Coomassie Blue Staining |
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5 | (1) |
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Basic Protocol 2 Nonammoniacal Silver Staining |
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6 | (1) |
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Alternate Protocol 2 Rapid Silver Staining |
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7 | (1) |
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Basic Protocol 3 Fluorescent Staining Using SYPRO Orange or Red |
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8 | (2) |
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Support Protocol: Photography of SYPRO Orange or Red Fluorescently Stained Gels |
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10 | (1) |
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11 | (1) |
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12 | (1) |
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13 | (1) |
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13 | (1) |
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14 | (1) |
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14 | |
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1 | (2) |
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3 | (1) |
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Southern and Northern Blotting |
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4 | (11) |
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15 | (4) |
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19 | (1) |
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20 | |
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8.2 Nucleic Acid Blotting: Southern and Northern |
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1 | (3) |
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4 | (1) |
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5 | (2) |
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7 | (1) |
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7 | (1) |
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Basic Protocol 1 Southern Blotting |
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7 | (6) |
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Basic Protocol 2 Northern Blotting |
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13 | (4) |
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Support Protocol: Assembling a Blotting Transfer Apparatus |
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17 | (1) |
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18 | (2) |
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20 | (1) |
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20 | (4) |
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24 | (1) |
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24 | (1) |
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24 | (1) |
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24 | |
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8.3 Protein Blotting: Immunoblotting |
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1 | (6) |
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7 | (1) |
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8 | (2) |
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10 | (1) |
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Basic Protocol 1 Protein Blotting with Semidry Systems |
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11 | (4) |
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Basic Protocol 2 Tank Transfer |
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15 | (3) |
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Alternate Protocol 1 Slot and Dot Blotting |
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18 | (1) |
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Support Protocol 1 Ponceau S Staining of Transferred Proteins |
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19 | (1) |
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Support Protocol 2 India Ink Staining of Transferred Proteins |
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19 | (1) |
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Support Protocol 3 Gold Staining of Transferred Proteins |
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19 | (1) |
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Support Protocol 4 Alkali Enhancement of Protein Staining |
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20 | (1) |
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Support Protocol 5 Fluorescent Protein Blot Staining of Transferred Proteins |
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20 | (2) |
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Support Protocol 6 Viewing and Photographing SYPRO Ruby-Stained Protein Blots |
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22 | (1) |
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Basic Protocol 3 Immunoprobing with Directly Conjugated Secondary Antibody |
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23 | (2) |
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Alternate Protocol 2 Immunoprobing with Avidin-Biotin Coupling to Secondary Antibody |
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25 | (1) |
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Basic Protocol 4 Visualization with Chromogenic Substrates |
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26 | (1) |
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Alternate Protocol 3 Visualization with Luminescent Substrates |
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27 | (2) |
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Alternate Protocol 4 Fluorescent Blot Preparation and Analysis |
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29 | (6) |
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35 | (2) |
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37 | (1) |
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37 | (2) |
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39 | (1) |
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39 | (1) |
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40 | |
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8.4 Labeling DNA and Preparing Probes |
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1 | (11) |
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12 | (1) |
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13 | (1) |
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13 | (1) |
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Basic Protocol 1 5' End-Labeling of DNA with T4 Polynucleotide Kinase |
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13 | (2) |
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Basic Protocol 2 Labeling DNA by Nick Translation |
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15 | (2) |
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Basic Protocol 3 Labeling DNA by Random Primed Synthesis |
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17 | (2) |
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Support Protocol: Purification of Labeled Probes Using Gel-Filtration Spin Columns |
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19 | (1) |
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20 | (1) |
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20 | (1) |
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20 | (1) |
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20 | (2) |
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22 | (1) |
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23 | |
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9.1 Conventional Light Microscopy |
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Parts of the Light Microscope |
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1 | (1) |
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1 | (3) |
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Basic Principles and Definitions |
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4 | (3) |
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Magnification Versus Resolution |
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7 | (2) |
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9 | (1) |
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Finding the Object to be Viewed |
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10 | (2) |
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Marking the Location of an Object: Secrets of the Microscope Stage |
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12 | (1) |
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A Quick Guide to Choosing from Various Optical Techniques |
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13 | (1) |
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Kohler Illumination: Secrets of the Substage Condenser |
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13 | (3) |
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16 | (3) |
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Dark-Field, Rheinberg, Polarized-Light, Phase-Contrast, and DIC Microscopy |
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19 | (9) |
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28 | (1) |
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29 | (1) |
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29 | (1) |
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29 | |
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9.2 Immunofluorescence Microscopy |
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1 | (6) |
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7 | (3) |
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10 | (1) |
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10 | (1) |
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Basic Protocol 1 Processing Fibroblasts |
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10 | (4) |
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Basic Protocol 2 Processing Tetrahymena Cells |
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14 | (1) |
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Alternate Protocol: Staining Cells Adhered to Poly-L-Lysine-Coated Coverslips |
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15 | (1) |
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Basic Protocol 3 Visualizing the Cells |
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16 | (3) |
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19 | (1) |
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20 | (1) |
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21 | (1) |
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22 | (1) |
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22 | (1) |
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23 | |
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10.1 Working with Enzymes |
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1 | (1) |
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1 | (5) |
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Handling Enzymes in the Laboratory |
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6 | (10) |
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Example of Setting Up an Enzymatic Reaction: Restriction Enzymes |
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16 | (5) |
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21 | (1) |
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21 | (1) |
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21 | |
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1 | (14) |
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15 | (4) |
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19 | (1) |
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20 | (1) |
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20 | (1) |
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Basic Protocol: Routine PCR |
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21 | (5) |
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Support Protocol 1 Using Temperature Gradients for Rapid Optimization of PCR Cycling Conditions |
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26 | (1) |
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Support Protocol 2 Titration of MgCl2 Concentration |
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26 | (1) |
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27 | (1) |
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28 | (1) |
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29 | (1) |
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29 | (3) |
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32 | (3) |
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35 | |
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1 | (4) |
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5 | (2) |
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7 | (19) |
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26 | (1) |
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26 | (1) |
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Basic Protocol 1 Synthesis of cDNA by Reverse Transcription |
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27 | (1) |
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Basic Protocol 2 Real-Time PCR Amplification and Analysis |
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28 | (3) |
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Support Protocol 1 Determination of Amplification Efficiency |
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31 | (1) |
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Support Protocol 2 Analyzing Results Using the Pfaffl Method to Calculate Fold Induction |
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32 | (2) |
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Support Protocol 3 Serial Dilution for Standard Curve |
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34 | (1) |
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35 | (1) |
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35 | (3) |
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38 | (1) |
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38 | (2) |
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40 | |
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1 | (4) |
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5 | (1) |
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5 | (4) |
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9 | (1) |
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Basic Protocol 1 Search a Nucleotide Database Using a Nucleotide Query: Nucleotide BLAST (BLASTN) |
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9 | (3) |
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Basic Protocol 2 Search a Protein Database Using a Protein Query: Protein BLAST (BLASTP) |
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12 | (1) |
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Basic Protocol 3 Search a Protein Database Using a Translated Nucleotide Query: BLASTX |
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13 | (2) |
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Basic Protocol 4 Searching a Translated Nucleotide Database Using a Protein or Nucleotide Query: TBLASTN and TBLASTX |
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15 | (1) |
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16 | (8) |
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24 | (1) |
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25 | (1) |
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26 | |
| Index |
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