分子生物学基础实验 双语教程PDF电子书下载

1实验室基本知识 1

1.1实验室安全 1

1.2如何正确使用实验室设备 3

1.2.1天平 3

1.2.2移液器 4

1.2.3离心（法） 6

1.2.4 pH计 10

1.2.5分光光度计 12

1.3溶液的灭菌与贮存 15

1.3.1溶液的灭菌 15

1.3.2生物溶液的贮存 15

2凝胶电泳 18

2.1介绍 18

2.1.1理论思考 18

2.1.2两种常见的凝胶介质 19

2.1.3灌制琼脂糖凝胶 21

2.1.4灌制聚丙烯酰胺凝胶 23

2.2核酸的凝胶电泳 25

2.2.1 DNA的琼脂糖凝胶电泳 25

2.2.2 DNA的聚丙烯酰胺凝胶电泳 27

2.2.3 RNA的琼脂糖凝胶电泳 28

2.2.4方案：DNA的琼脂糖凝胶电泳 31

2.2.5方案：RNA的甲醛凝胶电泳 32

2.2.6方案：RNA的乙二醛/DMSO凝胶电泳 32

2.3蛋白质的聚丙烯酰胺凝胶电泳 33

2.3.1介绍 33

2.3.2电泳缓冲液 34

2.3.3蛋白质在聚丙烯酰胺凝胶上的装载与电泳 34

2.3.4最佳电压、电泳时间和电源设置 36

3核酸的制备与质量控制 37

3.1基本原理 37

3.1.1裂解细胞 37

3.1.2核酸的纯化 37

3.1.3核酸的浓缩 38

3.2 DNA的提取 39

3.2.1 CTAB法小量制备植物基因组DNA 39

3.2.2盐析法分离高分子量DNA 42

3.2.3碱裂解法小量制备质粒DNA 45

3.3 RNA的提取 47

3.3.1基本原理 47

3.3.2控制RN ase活性 48

3.3.3胍—酸—酚抽提 49

3.3.4原核RNA的分离 52

3.4核酸的质量控制 54

3.4.1质量控制技术1： UV分光光度（测定）法与吸收比 54

3.4.2质量控制技术2：凝胶电泳 56

4核酸的限制性酶切分析 57

4.1 DNA的单一限制性核酸内切酶消化 57

4.2 DNA的多重酶切 60

5 PCR及其应用 61

5.1介绍 61

5.1.1 PCR如何运作？ 61

5.1.2 PC R反应混合液 62

5.1.3热稳定的DNA聚合酶 63

5.1.4热盖 63

5.1.5阳性与阴性对照 64

5.2标准PCR方案 65

5.3引物设计 67

5.4 PCR反应程序优化 69

5.5 PCR应用 71

5.5.1 LA PCR 71

5.5.2热启动PCR 71

5.5.3降落和上升PCR 72

5.5.4多重PCR 72

5.5.5反转录PCR 72

5.5.6定量和实时PCR 73

6从琼脂糖凝胶中回收DNA 75

6.1增加从琼脂糖凝胶中回收DNA效率的建议 75

6.2从低熔点琼脂糖凝胶中回收DNA：有机抽提 76

6.3从琼脂糖凝胶中电洗脱DNA 79

6.4用硅基膜离心柱从琼脂糖凝胶中回收DNA 81

7转化 84

7.1介绍 84

7.2大肠杆菌的CaC12转化 85

7.2.1方法1：传统方案 85

7.2.2方法2：革新方案 88

7.3大肠杆菌的电穿孔转化 90

8 DNA重组 95

8.1介绍 95

8.2载体与插入片段的准备 96

8.3连接 97

8.3.1介绍 97

8.3.2.标准应用——DNA的连接 98

8.4转化 99

8.5重组子的筛选 99

8.5.1插入失活 99

8.5.2菌液PCR 102

8.5.3限制（性酶切）图 103

9构建DNA文库 105

9.1基因组文库 106

9.1.1构建一个基因组文库 106

9.1.2载体的选择 106

9.1.3基因组文库的评估 107

9.1.4文库的生长与贮存 108

9.2 cDNA文库 108

9.2.1 mRNA的分离 109

9.2.2 cDNA的合成 109

9.2.3方案：使用试剂盒构建cDNA文库 113

10外源基因的表达 119

10.1介绍 119

10.2无细胞蛋白质表达系统/体外蛋白质表达系统 119

10.3体内的蛋白质表达系统 121

10.3.1大肠杆菌表达系统 121

10.3.2方案：克隆基因在大肠杆菌中的诱导表达与SDS-PAGE分析 125

11印迹技术 128

11.1技术指导 128

11.1.1印迹膜的选择 128

11.1.2转移方法 130

11.2核酸印迹 132

11.2.1核酸印迹概况 132

11.2.2固定 132

11.2.3核酸的标记与检测 133

11.2.4 DNA印迹方案 135

11.2.5 RNA印迹方案 139

11.3蛋白质印迹 142

11.3.1蛋白质印迹概况 142

11.3.2蛋白质印迹方案 146

附录A：常用的贮存溶液 150

附录B：培养基与抗生素 153

附录C：大分子制备与纯化试剂 155

附录D：核酸电泳与印迹的试剂和溶液 158

附录E：蛋白质电泳与印迹的试剂和溶液 160

1 General Laboratory Information 163

1.1 Safety in the Laboratory/Laboratory Safety 163

1.2 How to Properly Use Laboratory Equipment 166

1.2.1 Balances 166

1.2.2 Pipettors 167

1.2.3 Centrifugation 170

1.2.4 pH Meters 175

1.2.5 Spectrophotometers 178

1.3 Sterilization and Storage of Solutions 181

1.3.1 Sterilization of Solutions 181

1.3.2 Storage of Biological Solutions 181

2 Gel Electrophoresis 186

2.1 Introduction 186

2.1.1 Theoretical Considerations 186

2.1.2 Two Common Types of Gel Matrix 188

2.1.3 Casting Agarose Gel 190

2.1.4 Casting Polyacrylamide Gel 192

2.2 Nucleic Acid Gel Electrophoresis 194

2.2.1 Agarose Gel Electrophoresis of DNA 195

2.2.2 Polyacrylamide Gel Electrophoresis of DNA 197

2.2.3 Agarose Gel Electrophoresis of RNA 199

2.2.4 Protocol： Agarose Gel Electrophoresis of DNA 202

2.2.5 Protocol： Formaldehyde Electrophoresis of RNA 203

2.2.6 Protocol： Glyoxal/DMSO Electrophoresis of RNA 204

2.3 Protein Polyacrylamide Gel Electrophoresis 204

2.3.1 Introduction 204

2.3.2 Buffers for Electrophoresis 205

2.3.3 Loading and Running Proteins on Polyacrylamide Gels 205

2.3.4 Optimal Voltage， Running Times and Power Settings 207

3 Preparation and Quality Control of Nucleic Acid 209

3.1 Rationale 209

3.1.1 Lysising Cells 209

3.1.2 Purification of Nucleic Acid 209

3.1.3 Concentration of Nucleic Acid 211

3.2 Extraction of DNA 211

3.2.1 Small-scale Preparation of Plant Genomic DNA Using CTAB 211

3.2.2 Isolation of High-molecular-weight DNA by Salting-Out 215

3.2.3 Preparation of Plasmid DNA—Alkaline Lysis Minprep 218

3.3 Extraction of RNA 221

3.3.1 Rationale 221

3.3.2 Controlling RNase Activity 222

3.3.3 Guanidinium-Acid-Phenol Extraction 224

3.3.4 Isolation of Prokaryotic RNA 227

3.4 Quality Control of Nucleic Acid 230

3.4.1 Quality Control Technique 1： UV Spectrophotometry and Absorption Ratios 230

3.4.2 Quality Control Technique 2： Gel Electrophoresis 232

4 Restriction Analysis of Nucleic acid 233

4.1 Digesting DNA with a Single Restriction Endonuclease 233

4.2 Digesting DNA with Multiple Restriction Endonuclease 236

5 PCR and Its Application 237

5.1 Introduction 237

5.1.1 How does PCR work？ 237

5.1.2 The PCR Reaction Mix 238

5.1.3 Thermostable DNA Polymerases 239

5.1.4 Heated Lid 240

5.1.5 Positive and Negative Controls 240

5.2 Protocol of Standard PCR 242

5.3 Primers Design 244

5.4 Optimization of the PCR Reaction Procedures 246

5.5 PCR Applications 250

5.5.1 LA PCR 250

5.5.2 Hot Start PCR 251

5.5.3 Touchdown and Touch-up PCR 251

5.5.4 Multiplex PCR 252

5.5.5 Reverse-transcriptase PCR 252

5.5.6 Quantitative and Real-time PCR 253

6 Recovery of DNA from Agarose Gel 256

6.1 Tips for Increasing DNA Recovery Efficiency from Agarose Gels 256

6.2 Recovery of DNA from Low-melting-temperature Agarose Gels： Organic Extraction 257

6.3 Electroelution of DNA from Agarose Gels 260

6.4 Recovery of DNA from Agarose Gel Using Silica Membrane Spin Columns 262

7 Transformation 266

7.1 Introduction 266

7.2 Transformation of Escherichia coli Using CaCl2 267

7.2.1 Protocol 1：Conventional Scheme 267

7.2.2 Protocol 2：Innovation Scheme 270

7.3 Transformation of E.coli by Electroporation 272

8 DNA Recombination 277

8.1 Introduction 277

8.2 Preparation of Vector and Insert 278

8.3 Ligation 279

8.3.1 Introduction 279

8.3.2 Standard Application—Ligation of DNA 281

8.4 Transformation 282

8.5 Screening for Recombinants 282

8.5.1 I nserti onal Inactivation 282

8.5.2 PCR Amplification of Inserts from Bacterial Cultures 285

8.5.3 Restriction Mapping 287

9 Constructing DNA Libraries 289

9.1 Genomic Library 290

9.1.1 Construction of a Genomic Library 290

9.1.2 Choice of Vectors 290

9.1.3 Evaluation of A Genomic Library 292

9.1.4 Growing and Storing Libraries 292

9.2 cDNA Library 293

9.2.1 Isolation of mRNA 294

9.2.2 cDNA Synthesis 294

9.2.3 Protocol： Construction of A cDNA Library Using Kits 299

10 Foreign Gene Expressions/Recombinant Gene Expressions 305

10.1 Introduction 305

10.2 Cell-Free Protein Expression Systems/In vitro Protein Expression Systems 305

10.3 In vivo Protein Expression Systems 306

10.3.1 E.coli Expression System 306

10.3.2 Protocol： Expression of Cloned Genes in E.coli Using IPTG as Inducer and SDS-PAGE Analysis 312

11 Blotting Technology 316

11.1 Technology Guide 317

11.1.1 Choice of Blotting Membrane 317

11.1.2 Transfer Methods 319

11.2 Nucleic Acid Blotting 321

11.2.1 Overview of Nucleic Acid Blotting 321

11.2.2 Immobilization/Fixation 322

11.2.3 Nucleic Acid Labeling and Detection 323

11.2.4 Southern Blotting Protocol 326

11.2.5 Northern Blotting Protocol 331

11.3 Western Blotting 333

11.3.1 Overview of Western Blotting 334

11.3.2 Western Blotting Protocol 340

Appendix A： General Stock Solutions 344

Appendix B： Media and Antibiotics 347

Appendix C： Reagents and Solutions for Preparation and Purification of Macromolecular 349

Appendix D： Reagents and Solutions for Nucleic Acid Electrophoresis and Blotting 352

Appendix E： Reagents and Solutions for Protein Electrophoresis and Blotting 354

参考文献（References） 357