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| 082 | _ae-book (FST) | ||
| 100 | _aHarisha, S. | ||
| 110 | _b | ||
| 245 | _aBiotechnology procedures and experiments hand book / | ||
| 260 |
_aNew Delhi, India : _bInfinity science press, _c2007. |
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| 520 | _aCONTENTS Chapter 1. General Instruction and Laboratory Methods 1 General Instruction 1 General Laboratory Methods 2 Chapter 2. Tools and Techniques in Biological Studies 9 Spectrophotometry 9 Electrophoresis 13 Column Chromatography 18 pH Meter 22 Centrifugation 24 Ultrasonic Cell Disruption 29 Conductivity Meter 32 Radioactive Tracers 32 Autoradiography 37 Photography 39 Statistics 41 Graphs 44 Computers 47 Chapter 3. Biochemistry 51 Carbohydrates 51 Exercise 1. Qualitative Tests 52 Exercise 2. Qualitative Analysis 54 Exercise 3. Estimate the Amount of Reducing Sugars 56 Exercise 4. Estimation of Reducing Sugar by Somogyi’s Method 59 Exercise 5. Estimation of Sugar by Folin-Wu Method 60 vii viii CONTENTS Exercise 6. Estimation of Sugar by Hagedorn-Jenson Method 61 Exercise 7. Estimation of Reducing Sugars by the Dinitro Salicylic Acid (DNS) Method 62 Exercise 8. Determination of Blood Glucose by Hagedorn-Jenson Method 63 Exercise 9. Determining Blood Sugar by Nelson and Somogyi’s Method 65 Exercise 10. Determination of Blood Glucose by the O-Toluidine Method 66 Exercise 11. Estimation of Protein by the Biuret Method 67 Exercise 12. Estimation of Protein by the FC-Method 68 Exercise 13. Protein Assay by Bradford Method 69 Exercise 14. Estimation of Protein by the Lowry Protein Assay 70 Exercise 15. Biuret Protein Assay 71 Exercise 16. Estimation of DNA by the Diphenylamine Method 72 Exercise 17. Estimation of RNA by the Orcinol Method 73 Chapter 4. Enzymology 75 Enzymes 75 Exercise 1. Demonstrating the Presence of Catalase in Pig’s Liver 83 Exercise 2. Determining the Optimum pH for Trypsin 84 Exercise 3. Extraction of Tyrosinase 85 Exercise 4. Preparation of Standard Curve 87 Exercise 5. Enzyme Concentration 89 Exercise 6. Effects of pH 90 Exercise 7. Effects of Temperature 91 Exercise 8. Computer Simulation of Enzyme Activity 92 Exercise 9. Kinetic Analysis 93 Exercise 10. Determination of Km and Vmax 94 Exercise 11. Addition of Enzyme Inhibitors 95 Exercise 12. Protein Concentration/Enzyme Activity 97 Exercise 13. Studying the Action and Activity of Amylase on Starch Digestion 98 Exercise 14. Determination of the Effect of pH on the Activity of Human Salivary a-Amylase 99 CONTENTS ix Exercise 15. Determining the Effect of Temperature on the Activity of Human Salivary a-Amylase 101 Exercise 16. Construction of the Maltose Calibration Curve 102 Chapter 5. Electrophoresis 103 Electrophoresis 103 Exercise 1. Preparation of SDS-Polyacrylamide Gels 108 Exercise 2. Separation of Protein Standards: SDS-PAGE 110 Exercise 3. Coomassie Blue Staining of Protein Gels 111 Exercise 4. Silver Staining of Gels 112 Exercise 5. Documentation 114 Exercise 6. Western Blots 114 Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) 117 Preparation of Polyacrylamide Gels 119 Agarose Gel Electrophoresis 121 Agarose Gel Electrophoresis 123 Chapter 6. Microbiology 125 Introduction 125 The Microscopy 127 Exercise 1. The Bright Field Microscope 134 Exercise 2. Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms 137 Exercise 3. Cell Size Measurements: Ocular and Stage Micrometers 146 Exercise 4. Measuring Depth 147 Exercise 5. Measuring Area 148 Exercise 6. Cell Count by Hemocytometer or Measuring Volume 149 Exercise 7. Measurement of Cell Organelles 152 Exercise 8. Use of Darkfield Illumination 152 Exercise 9. The Phase Contrast Microscope 153 Exercise 10. The Inverted Phase Microscope 155 Aseptic Technique and Transfer of Microorganisms 155 Control of Microorganisms by using Physical Agents 163 x CONTENTS Control of Microorganisms by using Disinfectants and Antiseptics 172 Control of Microorganisms by using Antimicrobial Chemotherapy 180 Isolation of Pure Cultures from a Mixed Population 191 Bacterial Staining 201 Direct Stain and Indirect Stain 206 Gram Stain and Capsule Stain 211 Endospore Staining and Bacterial Motility 215 Enumeration of Microorganisms 221 Biochemical Test for Identification of Bacteria 233 Triple Sugar Iron Test 241 Starch Hydrolysis Test (II Method) 242 Gelatin Hydrolysis Test 244 Catalase Test 245 Oxidase Test 247 IMVIC Test 249 Extraction of Bacterial DNA 257 Medically Significant Gram–Positive Cocci (GPC) 259 Protozoans, Fungi, and Animal Parasites 263 The Fungi, Part 1–The Yeasts 264 Performance Objectives 267 The Fungi, Part 2—The Molds 269 Viruses: The Bacteriophages 274 Serology, Part 1–Direct Serologic Testing 279 Serology, Part 2–Indirect Serologic Testing 289 Chapter 7. Cell Biology and Genetics 299 Cell Cycles 299 Meiosis in Flower Buds of Allium Cepa-Acetocarmine Stain 306 Meiosis in Grasshopper Testis (Poecilocerus Pictus) 309 Mitosis in Onion Root Tip (Allium Cepa) 311 Differential Staining of Blood 313 Buccal Epithelial Smear and Barr Body 315 Vital Staining of DNA and RNA in Paramecium 316 CONTENTS xi Induction of Polyploidy 317 Mounting of Genitalia in Drosophila Melanogaster 318 Mounting of Genitalia in the Silk Moth Bombyx Mori 319 Mounting of the Sex Comb in Drosophila Melanogaster 320 Mounting of the Mouth Parts of the Mosquito 321 Normal Human Karyotyping 322 Karyotyping 322 Black and White Film Development and Printing for Karyotype Analysis 324 Study of Drumsticks in the Neutrophils of Females 327 Study of the Malaria Parasite 328 Vital Staining of DNA and RNA in Paramecium 330 Sex-Linked Inheritance in Drosophila Melanogaster 331 Preparation of Somatic Chromosomes from Rat Bone Marrow 333 Chromosomal Aberrations 334 Study of Phenocopy 336 Study of Mendelian Traits 337 Estimation of Number of Erythrocytes [RBC] in Human Blood 338 Estimation of Number of Leucocytes (WBC) in Human Blood 340 Culturing Techniques and Handling of Flies 342 Life Cycle of the Mosquito (Culex Pipiens) 343 Life Cycle of the Silkworm (Bombyx Mori) 344 Vital Staining of Earthworm Ovary 346 Culturing and Observation of Paramecium 347 Culturing and Staining of E.coli (Gram’s Staining) 348 Breeding Experiments in Drosophila Melanogaster 349 Preparation of Salivary Gland Chromosomes 354 Observation of Mutants in Drosophila Melanogaster 355 ABO Blood Grouping and Rh Factor in Humans 356 Determination of Blood Group and Rh Factor 357 Demonstration of the Law of Independent Assortment 358 Demonstration of Law of Segregation 360 xii CONTENTS Chapter 8. Molecular Biology 363 The Central Dogma 363 Exercise 1. Protein Synthesis in Cell Free Systems 364 Chromosomes 366 Exercise 2. Polytene Chromosomes of Dipterans 367 Exercise 3. Salivary Gland Preparation (Squash Technique) 368 Exercise 4. Extraction of Chromatin 369 Exercise 5. Chromatin Electrophoresis 371 Exercise 6. Extraction and Electrophoresis of Histones 372 Exercise 7. Karyotype Analysis 375 Exercise 8. In Situ Hybridization 378 Exercise 9. Culturing Peripheral Blood Lymphocytes 380 Exercise 10. Microslide Preparation of Metaphases for In-Situ Hybridization 382 Exercise 11. Staining Chromosomes (G-Banding) 384 Nucleic Acids 386 Exercise 12. Extraction of DNA from Bovine Spleen 387 Exercise 13. Purification of DNA 388 Exercise 14. Characterization of DNA 389 Exercise 15. DNA-Dische Diphenylamine Determination 390 Exercise 16. Melting Point Determination 391 Exercise 17. CsCl-Density Separation of DNA 393 Exercise 18. Phenol Extraction of rRNA (Rat liver) 394 Exercise 19. Spectrophotometric Analysis of rRNA 395 Exercise 20. Determination of Amount of RNA by the Orcinol Method 396 Exercise 21. Sucrose Density Fractionation 397 Exercise 22. Nucleotide Composition of RNA 398 Isolation of Genomic DNA—DNA Extraction Procedure 400 Exercise 23. Isolation of Genomic DNA from Bacterial Cells 401 Exercise 24. Preparation of Genomic DNA from Bacteria 404 Exercise 25. Extraction of Genomic DNA from Plant Source 405 Exercise 26. Extraction of DNA from Goat Liver 407 Exercise 27. Isolation of Cotton Genomic DNA from Leaf Tissue 408 CONTENTS xiii Exercise 28. Arabidopsis Thaliana DNA Isolation 411 Exercise 29. Plant DNA Extraction 412 Exercise 30. Phenol/Chloroform Extraction of DNA 414 Exercise 31. Ethanol Precipitation of DNA 414 Exercise 32. Isolation of Mitochondrial DNA 415 Exercise 33. Isolation of Chloroplast DNA 417 Exercise 34. DNA Extraction of Rhizobium (CsCl Method) 417 Exercise 35. Isolation of Plasmids 418 Exercise 36. RNA Isolation 422 Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity 422 Exercise 37. RNA Extraction Method for Cotton 423 Exercise 38. Isolation of RNA from Bacteroids 424 Exercise 39. Isolation of RNA from Free-Living Rhizobia 425 Estimation of DNA purity and Quantification 426 Exercise 40. Fungal DNA Isolation 427 Exercise 41. Methylene Blue DNA Staining 429 Exercise 42. Transformation 431 Blotting Techniques—Southern, Northern, Western Blotting 433 Preparing the Probe 440 Exercise 43. Southern Blotting (First Method) 444 Exercise 44. Southern Blotting (Second Method) 446 Exercise 45. Western Blotting 447 Exercise 46. Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies 450 Southern Blot 452 Exercise 47. Southern Analysis of Mouse Toe/Tail DNA 453 Exercise 48. Northern Blotting 454 Restriction Digestion Methods—Restriction Enzyme Digests 457 Exercise 49. Restriction Digestion of Plasmid, Cosmid, and Phage DNAs 460 xiv CONTENTS Exercise 50. Manual Method of Restriction Digestion of Human DNA 461 Exercise 51. Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs 463 Exercise 52. Restriction Enzyme Digestion of DNA 465 Exercise 53. Electroelution of DNA Fragments from Agarose into Dialysis Tubing 466 Exercise 54. Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose 469 Exercise 55. Ligation of Insert DNA to Vector DNA 471 PCR Methods (Polymerase Chain Reaction) 472 Exercise 56. Polymerase Chain Reaction 475 Exercise 57. DNA Amplification by the PCR Method 476 Chapter 9. Tissue Culture Techniques 477 Tissue Culture Methods 477 Plant Tissue Culture 487 Plant Tissue Culture 491 Many Dimensions of Plant Tissue Culture Research 493 What is Plant Tissue Culture? 496 Uses of Plant Tissue Culture 497 Plant Tissue Culture demonstration by Using Somaclonal Variation to Select for Disease Resistance 498 Demonstration of Tissue Culture for Teaching 499 Preparation of Plant Tissue Culture Media 502 Plant Tissue Culture Media 505 Preparation of Protoplasts 512 Protoplast Isolation, Culture, and Fusion 514 Agrobacterium Culture and Agrobacterium— Mediated transformation 518 Isolation of Chloroplasts from Spinach Leaves 520 Preparation of Plant DNA using CTAB 522 Suspension Culture and Production of Secondary Metabolites 525 Protocols for Plant Tissue Culture 529 Sterile Methods in Plant Tissue Culture 535 CONTENTS xv Media for Plant Tissue Culture 538 Safety in Plant Tissue Culture 543 Preparation of Media for Animal Cell Culture 544 Aseptic Technique 546 Culture and Maintenance of Cell Lines 550 Trypsinizing and Subculturing Cells from a Monolayer 557 Cellular Biology Techniques 558 In Vitro Methods 574 Human Cell Culture Methods 577 Chapter 10. Tests 595 Starch Hydrolysis Test 595 Gelatin Hydrolysis Test 597 Catalase Test 598 Oxidase Test 599 Appendix A (Units and Measures) 603 Appendix B (Chemical Preparations) 617 Appendix C (Reagents Required for Tissue Culture Experiments) 645 Appendix D (Chemicals Required for Microbiology Experiments) 671 Appendix E (About the CD-ROM) 689 Index 691 | ||
| 700 | _aBamforth, Charles W | ||
| 942 |
_2ddc _cEB |
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| 999 |
_c8914 _d8914 |
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