分子生物学基础实验 双语教程2025|PDF|Epub|mobi|kindle电子书版本百度云盘下载

分子生物学基础实验 双语教程PDF格式电子书版下载

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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DNA39

3.2.2盐析法分离高分子量DNA42

3.2.3碱裂解法小量制备质粒DNA45

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 PCR71

5.5.2热启动PCR71

5.5.3降落和上升PCR72

5.5.4多重PCR72

5.5.5反转录PCR72

5.5.6定量和实时PCR73

6从琼脂糖凝胶中回收DNA75

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DNA79

6.4用硅基膜离心柱从琼脂糖凝胶中回收DNA81

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PCR102

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 Information163

1.1 Safety in the Laboratory/Laboratory Safety163

1.2 How to Properly Use Laboratory Equipment166

1.2.1 Balances166

1.2.2 Pipettors167

1.2.3 Centrifugation170

1.2.4 pH Meters175

1.2.5 Spectrophotometers178

1.3 Sterilization and Storage of Solutions181

1.3.1 Sterilization of Solutions181

1.3.2 Storage of Biological Solutions181

2 Gel Electrophoresis186

2.1 Introduction186

2.1.1 Theoretical Considerations186

2.1.2 Two Common Types of Gel Matrix188

2.1.3 Casting Agarose Gel190

2.1.4 Casting Polyacrylamide Gel192

2.2 Nucleic Acid Gel Electrophoresis194

2.2.1 Agarose Gel Electrophoresis of DNA195

2.2.2 Polyacrylamide Gel Electrophoresis of DNA197

2.2.3 Agarose Gel Electrophoresis of RNA199

2.2.4 Protocol： Agarose Gel Electrophoresis of DNA202

2.2.5 Protocol： Formaldehyde Electrophoresis of RNA203

2.2.6 Protocol： Glyoxal/DMSO Electrophoresis of RNA204

2.3 Protein Polyacrylamide Gel Electrophoresis204

2.3.1 Introduction204

2.3.2 Buffers for Electrophoresis205

2.3.3 Loading and Running Proteins on Polyacrylamide Gels205

2.3.4 Optimal Voltage， Running Times and Power Settings207

3 Preparation and Quality Control of Nucleic Acid209

3.1 Rationale209

3.1.1 Lysising Cells209

3.1.2 Purification of Nucleic Acid209

3.1.3 Concentration of Nucleic Acid211

3.2 Extraction of DNA211

3.2.1 Small-scale Preparation of Plant Genomic DNA Using CTAB211

3.2.2 Isolation of High-molecular-weight DNA by Salting-Out215

3.2.3 Preparation of Plasmid DNA—Alkaline Lysis Minprep218

3.3 Extraction of RNA221

3.3.1 Rationale221

3.3.2 Controlling RNase Activity222

3.3.3 Guanidinium-Acid-Phenol Extraction224

3.3.4 Isolation of Prokaryotic RNA227

3.4 Quality Control of Nucleic Acid230

3.4.1 Quality Control Technique 1： UV Spectrophotometry and Absorption Ratios230

3.4.2 Quality Control Technique 2： Gel Electrophoresis232

4 Restriction Analysis of Nucleic acid233

4.1 Digesting DNA with a Single Restriction Endonuclease233

4.2 Digesting DNA with Multiple Restriction Endonuclease236

5 PCR and Its Application237

5.1 Introduction237

5.1.1 How does PCR work？237

5.1.2 The PCR Reaction Mix238

5.1.3 Thermostable DNA Polymerases239

5.1.4 Heated Lid240

5.1.5 Positive and Negative Controls240

5.2 Protocol of Standard PCR242

5.3 Primers Design244

5.4 Optimization of the PCR Reaction Procedures246

5.5 PCR Applications250

5.5.1 LA PCR250

5.5.2 Hot Start PCR251

5.5.3 Touchdown and Touch-up PCR251

5.5.4 Multiplex PCR252

5.5.5 Reverse-transcriptase PCR252

5.5.6 Quantitative and Real-time PCR253

6 Recovery of DNA from Agarose Gel256

6.1 Tips for Increasing DNA Recovery Efficiency from Agarose Gels256

6.2 Recovery of DNA from Low-melting-temperature Agarose Gels： Organic Extraction257

6.3 Electroelution of DNA from Agarose Gels260

6.4 Recovery of DNA from Agarose Gel Using Silica Membrane Spin Columns262

7 Transformation266

7.1 Introduction266

7.2 Transformation of Escherichia coli Using CaCl2267

7.2.1 Protocol 1：Conventional Scheme267

7.2.2 Protocol 2：Innovation Scheme270

7.3 Transformation of E.coli by Electroporation272

8 DNA Recombination277

8.1 Introduction277

8.2 Preparation of Vector and Insert278

8.3 Ligation279

8.3.1 Introduction279

8.3.2 Standard Application—Ligation of DNA281

8.4 Transformation282

8.5 Screening for Recombinants282

8.5.1 I nserti onal Inactivation282

8.5.2 PCR Amplification of Inserts from Bacterial Cultures285

8.5.3 Restriction Mapping287

9 Constructing DNA Libraries289

9.1 Genomic Library290

9.1.1 Construction of a Genomic Library290

9.1.2 Choice of Vectors290

9.1.3 Evaluation of A Genomic Library292

9.1.4 Growing and Storing Libraries292

9.2 cDNA Library293

9.2.1 Isolation of mRNA294

9.2.2 cDNA Synthesis294

9.2.3 Protocol： Construction of A cDNA Library Using Kits299

10 Foreign Gene Expressions/Recombinant Gene Expressions305

10.1 Introduction305

10.2 Cell-Free Protein Expression Systems/In vitro Protein Expression Systems305

10.3 In vivo Protein Expression Systems306

10.3.1 E.coli Expression System306

10.3.2 Protocol： Expression of Cloned Genes in E.coli Using IPTG as Inducer and SDS-PAGE Analysis312

11 Blotting Technology316

11.1 Technology Guide317

11.1.1 Choice of Blotting Membrane317

11.1.2 Transfer Methods319

11.2 Nucleic Acid Blotting321

11.2.1 Overview of Nucleic Acid Blotting321

11.2.2 Immobilization/Fixation322

11.2.3 Nucleic Acid Labeling and Detection323

11.2.4 Southern Blotting Protocol326

11.2.5 Northern Blotting Protocol331

11.3 Western Blotting333

11.3.1 Overview of Western Blotting334

11.3.2 Western Blotting Protocol340

Appendix A： General Stock Solutions344

Appendix B： Media and Antibiotics347

Appendix C： Reagents and Solutions for Preparation and Purification of Macromolecular349

Appendix D： Reagents and Solutions for Nucleic Acid Electrophoresis and Blotting352

Appendix E： Reagents and Solutions for Protein Electrophoresis and Blotting354

参考文献（References）357