Molecular biology and biotechnology: basic experimental protocols
Book Details
Molecular Biology and Biotechnology: basic experimental protocols is a compilation of methods and techniques commonly used in biomedical and biotechnological studies. The book aims to provide ample support to both students and faculty while conducting practical lessons. Four sections are covered in this book—Genomics, Proteomics, Quantitative Biochemistry, and Bioinformatics. A concise introductory note accompanies each protocol/method described for better comprehension. Every topic discussed is supported by actual methods and their expected results, and is accompanied by relevant questions.
Key Features
- Ready design of basic experiments in biomedical and biotechnology sciences.
- Extensive coverage of basic equipment used in biomedical and biotechnological studies.
- Concise information on each subject, with focus on practical lessons.
Target Audience
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Graduate students, Postgraduate students, Technicians of Biomedical Science, Biotechnology
Table of Contents
SECTION I: GENOMICS
1. Initial Characterization of DNA
1.1 Mammalian High Molecular Weight DNA Extraction
1.2 Spectrophotometric Analysis of DNA
1.3 Agarose Gel Electrophoretic Analysis of DNA
1.4 Restriction Mapping of DNA
2. DNA Probe Preparation, Southern Blotting, and Hybridization
2.1 Oligonucleotides or Gene Probe Design
2.2 Labelling DNA Gene Probes
2.3 Southern Blotting of DNA and Hybridization
3. RFLP and SSCP Analysis of DNA
3.1 Restriction Fragment Length Polymorphism
3.2 Single-strand Conformation Polymorphism
4. Protein–DNA Interaction
4.1 DNase Footprinting of DNA
4.2 Gel Mobility Shift Assay
5. Plasmid DNA Analysis
5.1 Preparation and Maintenance of Microbial Culture
5.2 Plasmid and Chromosomal DNA Preparation from E. coli
5.3 Analysis of Plasmid and Chromosomal DNA on AgaroseGel Electrophoresis
5.4 Transformation of E. coli with Plasmid DNA
5.5 DNA Ligation and E. coli Transformation
6. Polymerase Chain Reaction
6.1 Amplification of DNA by PCR and Analysis on AgaroseGel Electrophoresis
6.2 Cloning PCR Products
6.3 DNA Sequencing
7. RNA Analysis Techniques
7.1 Total RNA Isolation from Tissue or Cells
7.2 Spectrophotometric Analysis of RNA
7.3 RNA Agarose Gel Electrophoresis
7.4 Messenger RNA Extraction from Total RNA
7.5 In Vitro Protein Synthesis
8. Gene or mRNA Expression in Cells/Tissues
8.1 Northern Blotting of RNA and Hybridization
8.2 Reverse Transcriptase-Polymerase Chain Reaction
8.3 Differential Display Polymerase Chain Reaction
8.4 In Situ Hybridization
9. Transcription Activity In Vivo
9.1 Nuclear Run-on Transcription
9.2 Reporter Gene Transcription
10. Apoptosis Analysis
10.1 DNA Ladder
10.2 TUNEL Assay
10.3 Comet Assay
SECTION II: PROTEOMICS
11. Initial Preparations for Protein Analysis
11.1 Buffers and Additional Components
11.2 Spectroscopic Analysis of Chromophores
11.3 Tissue and their Subcellular Fractionation
12. Quantitative Determination of Proteins
12.1 Lowry (Folin–Ciocalteu) Method
12.2 Bradford Method
12.3 Direct Spectrophotometric Method
13. Protein Separation and Analysis
13.1 Ion-exchange Chromatography
13.2 Gel Filtration Chromatography
13.3 Electrophoretic Analysis
13.4 Staining of Gels after Electrophoresis
14. Electroblotting, Dot/Slot Blotting, and Electroelution
14.1 Electroblotting
14.2 Dot/Slot Blotting
14.3 Staining Methods of Proteins on Electroblots
14.4 Electroelution of Proteins from Gels
14.5 Sample Preparation for Amino Acid Sequencing
15. Polyclonal Antibodies Production and Immunoanalysis
15.1 Production of Antibodies
15.2 Ouchterlony Double Immunodiffusion Method
15.3 Western Immunoblot
15.4 Enzyme-linked Immunosorbent Assay
15.5 Dot/Slot Immunoblot Assay
15.6 Immunoprecipitation
15.7 Immunohistochemistry
16. Protein–protein Interactions
16.1 Pull-Down Assay
16.2 Far-western Analysis
16.3 Co-immunoprecipitation
SECTION III: QUANTITATIVE BIOCHEMISTRY
17. Analysis of Biomolecules
17.1 Extraction and Estimation of DNA, RNA, and Protein from Tissues
17.2 Estimation of Oxidative Stress-related Biomolecules
18. Enzyme Kinetics and Enzyme Activity Analysis
18.1 Influence of Substrate Concentration on Enzyme Activity
18.2 Influence of Temperature on Enzyme Activity
18.3 Influence of pH on Enzyme Activity
18.4 Influence of Inhibitors on Enzyme Activity
18.5 Redox-active Enzyme Activity Estimations
19. Labelling of Proteins and its Uses
19.1 Radioiodination of Target Protein
19.2 In Vivo Receptor Assay Using 125I-labelled Protein
19.3 Radioactive Phosphorylation of Specific Protein (Tissue Protein Kinase Assay)
20. Statistical Analysis of Biochemical Data
20.1 t-Test
20.2 Analysis of Variance
SECTION IV: BIOINFORMATICS
21. Sequence Retrieval from Databases
21.1 Sequence Retrieval
21.2 Using Entrez Search Tags and Boolean Operators
21.3 FASTA Format
22. Sequence Similarity Search
22.1 Homologous Sequences
22.2 Pairwise Sequence Alignment
22.3 Multiple Sequence Alignment
23. Protein Sequence and Structure Analysis
23.1 Primary Protein Structure Analysis
23.2 Secondary Structure Prediction and Analysis
24. Nucleotide Sequence Analysis
24.1 Gene Prediction
24.2 Splice Site Prediction
24.3 Restriction Mapping of DNA
24.4 PCR Primer Designing
25. Molecular Phylogeny
25.1 Preparing Alignment File
25.2 Distance and Character-based Analysis
25.3 Using Tree Construction Matrices
25.4 Plotting Phylogenetic Tree
Keywords
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