CONTENTS 54. Solid-Phase Extraction for Preconcentration 45 5.5. Sample Preparation for Water Samples 5.7. Preparation of Sample Slurries for Direct AAS Analysis 5.8. Hydride Generation Methods 59. Colorimetric methods 5. 10. Metal Speciation 5. 10.1. Types of Speciation 5. 10.2. Speciation for Soils and Sediments 5.10.3. Sequential Schemes for Metals in Soil or sediment 5.10.4. Speciation for Metals in Plant Materials 5. 10.5. Speciation of Specific Elements 5.11. Contamination during Metal Analysis 5. 12. Safe Handling of Acids References SECTION B SAMPLE PREPARATION FOR NUCLEIC ACID ANALYSIS CHAPTER 6 SAMPLE PREPARATION N DNA ANALYSIS Satish parimoo and Bhama parimoe 6.1 dNA and its structure 6. 1. Physical and Chemical Properties of 6.1. 2. Isolation of dNa 6.2. Isolation of dna from bacteria 6.2. 1. Phenol Extraction and Precipitation of dNa 278 6.2.2. Removal of Contaminants from 63. Isolation of plasmid dna 6.3. 1. Plasmid DNA Preparation 6.3. 2. Purification of plasmid dNa 285 64. Genomic DNA Isolation from Yea
5.4. Solid-Phase Extraction for Preconcentration 245 5.5. Sample Preparation for Water Samples 248 5.6. Precipitation Methods 251 5.7. Preparation of Sample Slurries for Direct AAS Analysis 251 5.8. Hydride Generation Methods 252 5.9. Colorimetric Methods 254 5.10. Metal Speciation 255 5.10.1. Types of Speciation 257 5.10.2. Speciation for Soils and Sediments 258 5.10.3. Sequential Schemes for Metals in Soil or Sediment 259 5.10.4. Speciation for Metals in Plant Materials 260 5.10.5. Speciation of Specific Elements 262 5.11. Contamination during Metal Analysis 263 5.12. Safe Handling of Acids 264 References 264 SECTION B SAMPLE PREPARATION FOR NUCLEIC ACID ANALYSIS CHAPTER 6 SAMPLE PREPARATION IN DNA ANALYSIS 271 Satish Parimoo and Bhama Parimoo 6.1. DNA and Its Structure 271 6.1.1. Physical and Chemical Properties of DNA 274 6.1.2. Isolation of DNA 276 6.2. Isolation of DNA from Bacteria 278 6.2.1. Phenol Extraction and Precipitation of DNA 278 6.2.2. Removal of Contaminants from DNA 282 6.3. Isolation of Plasmid DNA 283 6.3.1. Plasmid DNA Preparation 284 6.3.2. Purification of Plasmid DNA 285 6.4. Genomic DNA Isolation from Yeast 287 xii contents
CONTENTS XIl 6.5. DNA from Mammalian Tissues 6.5.1. Blood 6.5.2. Tissues and tissue culture cells 66. DNA from plant Tissue 6.7. Isolation of Very High Molecular Weight DNA 290 6.8. DNA Amplification by Polymerase Chain Reaction 6.8.1. Starting a PCR Reaction 6.8.2. Isolation of dna from Small real World Samples for PCR 6.9. Assessment of Quality and Quantitation of 6.9.1. Precautions for Preparing DNA 6.9.2. Assessment of Concentration and 6.9.3. Storage of DNA References 299 CHAPTER T SAMPLE PREPARATION IN RNA ANALYSIS Bhama parimoo and satish Parimoo 7.1. RNA: Structure and Properties 7.1. 1. Types and Location of Various 7. 2. RNA Isolation: Basic Considerations 306 7. 2.1. Methods of Extraction and Isolation of RNa 7.3. Phenol Extraction and RNA Recovery Basic Principles 7.3.1. Examples of RNA Isolation Using Phenol extraction 310 74. Guanidinium Salt method 7.4.1. Examples of RNA Isolatic Guanidinium salts 7.5. Isolation of rna from nuclear and ytoplasmic Cellular Fractions 317
6.5. DNA from Mammalian Tissues 288 6.5.1. Blood 288 6.5.2. Tissues and Tissue Culture Cells 289 6.6. DNA from Plant Tissue 290 6.7. Isolation of Very High Molecular Weight DNA 290 6.8. DNA Amplification by Polymerase Chain Reaction 291 6.8.1. Starting a PCR Reaction 291 6.8.2. Isolation of DNA from Small RealWorld Samples for PCR 294 6.9. Assessment of Quality and Quantitation of DNA 296 6.9.1. Precautions for Preparing DNA 296 6.9.2. Assessment of Concentration and Quality 296 6.9.3. Storage of DNA 299 References 299 CHAPTER 7 SAMPLE PREPARATION IN RNA ANALYSIS 301 Bhama Parimoo and Satish Parimoo 7.1. RNA: Structure and Properties 301 7.1.1. Types and Location of Various RNAs 303 7.2. RNA Isolation: Basic Considerations 306 7.2.1. Methods of Extraction and Isolation of RNA 307 7.3. Phenol Extraction and RNA Recovery: Basic Principles 309 7.3.1. Examples of RNA Isolation Using Phenol Extraction 310 7.4. Guanidinium Salt Method 313 7.4.1. Examples of RNA Isolation Using Guanidinium Salts 313 7.5. Isolation of RNA from Nuclear and Cytoplasmic Cellular Fractions 317 contents xiii
XI CONTENTS 7. 6. Removal of DNA Contamination from 317 7. 7. Fractionation of RNA Using Chromatography Methods 318 7.7.1. Fractionation of Small RNa by HPLC 7.7.2. mRNA Isolation by Affinity Chromatography 7. 8. Isolation of rna from Small Numbers of Cells 7.9. In Vitro Synthesis of RNA 10. Assessment of Quality and quantitation of RNA 326 7. 11. Storage of RNA 328 CHAPTER 8 TECHNIQUES FOR THE EXTRACTION ISOLATION. AND PURIFICATION OF NUCLEIC ACIDS Mahesh Karwa and Somenath Mitra 8.1. Introduction 331 8.2. Methods of Cell Lysis 8.2. 1. Mechanical Methods of Cell Lysis 8.2. 2. Nonmechanical Methods of Cell 339 8. 3. Isolation of Nucleic Acids 8.3.1. Solvent Extraction and Precipitation 8.3.2. Membrane filtration 4. Chromatographic Methods for the Purification of Nucleic Acids 8.4. 1. Size-Exclusion Chromatography 8.4.2. Anion-Exchange Chromatography 348 8.4.3. Solid-Phase extraction 8.4. 4. Affinity Purification 5. Automated High-Throughput DNA Purification Systems 8.6. Electrophoretic Separation of Nucleic Acids 360
7.6. Removal of DNA Contamination from RNA 317 7.7. Fractionation of RNA Using Chromatography Methods 318 7.7.1. Fractionation of Small RNA by HPLC 318 7.7.2. mRNA Isolation by A‰nity Chromatography 319 7.8. Isolation of RNA from Small Numbers of Cells 323 7.9. In Vitro Synthesis of RNA 324 7.10. Assessment of Quality and Quantitation of RNA 326 7.11. Storage of RNA 328 References 329 CHAPTER 8 TECHNIQUES FOR THE EXTRACTION, ISOLATION, AND PURIFICATION OF NUCLEIC ACIDS 331 Mahesh Karwa and Somenath Mitra 8.1. Introduction 331 8.2. Methods of Cell Lysis 333 8.2.1. Mechanical Methods of Cell Lysis 335 8.2.2. Nonmechanical Methods of Cell Lysis 339 8.3. Isolation of Nucleic Acids 342 8.3.1. Solvent Extraction and Precipitation 344 8.3.2. Membrane Filtration 345 8.4. Chromatographic Methods for the Purification of Nucleic Acids 346 8.4.1. Size-Exclusion Chromatography 347 8.4.2. Anion-Exchange Chromatography 348 8.4.3. Solid-Phase Extraction 351 8.4.4. A‰nity Purification 352 8.5. Automated High-Throughput DNA Purification Systems 355 8.6. Electrophoretic Separation of Nucleic Acids 360 xiv contents
CONTENTS 8.6.1. Gel Electrophoresis for Nucleic Acids Purification 8.6. 2. Techniques for the Isolation of DNA from gels 8.7. Capillary Electrophoresis for Sequencing and Sizing 364 8. 8. Microfabricated Devices for Nucleic Acid Analys 366 8.8.1. Sample Preparation on Microchips 370 References 373 SECTION C SAMPLE PREPARATION IN MICROSCOPY AND SPECTROSCOPY CHAPTER 9 SAMIPLE PREPARATION FOR MICROSCOPIC AND SPECTROSCOPIC CHARACTERIZATION OF SOLID SURFACES AND FILMS Sharmila M. Mukhopadhyay 9.1. Introduction 377 9.1. 1. Microscopy of Solids 378 9. 1. 2. Spectroscopic Techniques for Solids 38 9.2. Sample Preparation for Microscopic Evaluation 9.2. 1. Sectioning and polishing 382 9.2.2. Chemical and Thermal Etching 9. 2.3. Sample Coating Techniques 9.3. Specimen Thinning for TEM Analysis 9.3.2. Reactive Ion Techniques 393 9.3.3. Chemical Polishing and metropol 9.3. 4. Tripod Polishing 9.3.5 Ultramicrotomy 398 9.3.6. Special Techniques and variations 9.4. Summary: Sample Preparation for Microscopy 400
8.6.1. Gel Electrophoresis for Nucleic Acids Purification 360 8.6.2. Techniques for the Isolation of DNA from Gels 362 8.7. Capillary Electrophoresis for Sequencing and Sizing 364 8.8. Microfabricated Devices for Nucleic Acids Analysis 366 8.8.1. Sample Preparation on Microchips 370 References 373 SECTION C SAMPLE PREPARATION IN MICROSCOPY AND SPECTROSCOPY CHAPTER 9 SAMPLE PREPARATION FOR MICROSCOPIC AND SPECTROSCOPIC CHARACTERIZATION OF SOLID SURFACES AND FILMS 377 Sharmila M. Mukhopadhyay 9.1. Introduction 377 9.1.1. Microscopy of Solids 378 9.1.2. Spectroscopic Techniques for Solids 381 9.2. Sample Preparation for Microscopic Evaluation 382 9.2.1. Sectioning and Polishing 382 9.2.2. Chemical and Thermal Etching 385 9.2.3. Sample Coating Techniques 387 9.3. Specimen Thinning for TEM Analysis 389 9.3.1. Ion Milling 391 9.3.2. Reactive Ion Techniques 393 9.3.3. Chemical Polishing and Electropolishing 394 9.3.4. Tripod Polishing 396 9.3.5. Ultramicrotomy 398 9.3.6. Special Techniques and Variations 399 9.4. Summary: Sample Preparation for Microscopy 400 contents xv
XVI CONTENTS 9.5. Sample Preparation for Surface Spectroscopy 9.5.1. Ion bombardment 9.5.2. Sample Heating 9.5.3. In Situ Abrasion and Scraping 9.5.4. In Situ Cleavage or Fracture Stage 9.5.5. Sample Preparation/Treatment Options for In Situ Reaction Studies 409 9.6. Summary: Sample Preparation for Surface Spectroscopy References 410 CHAPTER 10 SURFACE ENHANCEMENT BY SAMPLE AND SUBSTRATE PREPARATION TECHNIQUES IN RAMAN AND INFRARED SPECTROSCOPY 413 Zafar Igbal 10.1. Introduction 413 0.1.1. Raman effect 413 10.1.2. Fundamentals of Surface-Enhanced Raman Spectroscopy 415 10.1.3. Attenuated Total Reflection Infrared Spec 10.14. Fundamentals of surface-Enhanced Infrared Spectroscopy 10.2. Sample Preparation for SERS 10. 2.1. Electrochemical Techniques 10.2.2. Vapor Deposition and Chemical Preparation Techniques 10.2.3. Colloidal Sol Techniques 425 0.2.4. Nanoparticle Arrays and Gratings 10.3. Sample Preparation for SEIRA 10.4. Potential Applications INDEX
9.5. Sample Preparation for Surface Spectroscopy 402 9.5.1. Ion Bombardment 407 9.5.2. Sample Heating 408 9.5.3. In Situ Abrasion and Scraping 408 9.5.4. In Situ Cleavage or Fracture Stage 408 9.5.5. Sample Preparation/Treatment Options for In Situ Reaction Studies 409 9.6. Summary: Sample Preparation for Surface Spectroscopy 409 References 410 CHAPTER 10 SURFACE ENHANCEMENT BY SAMPLE AND SUBSTRATE PREPARATION TECHNIQUES IN RAMAN AND INFRARED SPECTROSCOPY 413 Zafar Iqbal 10.1. Introduction 413 10.1.1. Raman E¤ect 413 10.1.2. Fundamentals of Surface-Enhanced Raman Spectroscopy 415 10.1.3. Attenuated Total Reflection Infrared Spectroscopy 420 10.1.4. Fundamentals of Surface-Enhanced Infrared Spectroscopy 421 10.2. Sample Preparation for SERS 423 10.2.1. Electrochemical Techniques 423 10.2.2. Vapor Deposition and Chemical Preparation Techniques 424 10.2.3. Colloidal Sol Techniques 425 10.2.4. Nanoparticle Arrays and Gratings 427 10.3. Sample Preparation for SEIRA 431 10.4. Potential Applications 433 References 436 INDEX 439 xvi contents