Learning Objectives
4 objectives- Understand the fundamental principles and applications of key biochemical and molecular biology techniques.
- Develop the ability to analyze and interpret experimental data from spectrophotometry, gel electrophoresis, enzyme kinetics, and other lab methods.
- Gain practical knowledge of protein purification, cell culture, PCR, western blotting, and mass spectrometry techniques.
- Apply theoretical concepts to experimental design and troubleshooting in biochemical analysis.
Content Outline
PreviewUnit 2998: Advanced Biochemical and Molecular Biology Techniques
1. Spectrophotometry
1.1 Principles of Spectrophotometry
- Interaction of light with matter
- Absorbance and transmittance definitions
1.2 Beer-Lambert Law
- Mathematical expression and variables
- Limitations and assumptions
1.3 Applications
- Measuring concentration of biomolecules (proteins, nucleic acids)
- Enzyme activity assays
2. Gel Electrophoresis
2.1 Fundamentals
- Principle: separation based on size and charge
- Types of gels: agarose vs polyacrylamide
2.2 Agarose Gel Electrophoresis
- Preparation and running conditions
- Applications for nucleic acids
2.3 Polyacrylamide Gel Electrophoresis (PAGE)
- SDS-PAGE for proteins
- Native PAGE
- Visualization techniques (staining, fluorescent dyes)
3. Enzyme Kinetics
3.1 Enzyme-Catalyzed Reaction Rates
- Definition of reaction rate
- Factors influencing rate
3.2 Michaelis-Menten Kinetics
- Derivation and significance of Km and Vmax
- Lineweaver-Burk and other plots
3.3 Enzyme Inhibition
- Competitive, non-competitive, and uncompetitive inhibition
3.4 Factors Affecting Enzyme Activity
- pH, temperature, substrate concentration
4. Protein Purification
4.1 Overview
- Importance and goals of purification
- Purity assessment
4.2 Chromatography Techniques
- Affinity chromatography: principle and applications
- Ion exchange chromatography: cation and anion exchange
- Size exclusion chromatography: separation by molecular size
4.3 Additional Methods
- Dialysis and ultrafiltration
5. Polymerase Chain Reaction (PCR)
5.1 Principles
- DNA amplification and the role of Taq polymerase
- Thermal cycling steps: denaturation, annealing, extension
5.2 Types of PCR
- Conventional PCR
- Quantitative PCR (qPCR): principles and applications
- Reverse transcription PCR (RT-PCR) overview
5.3 Applications
- Molecular diagnostics
- Genetic analysis
6. Western Blotting
6.1 Overview
- Purpose of western blotting
6.2 Procedure
- Protein separation by gel electrophoresis
- Transfer to membrane (nitrocellulose or PVDF)
- Blocking and antibody incubation
- Detection methods (chemiluminescence, fluorescence)
6.3 Data Analysis
- Interpretation of bands
- Quantification techniques
7. Mass Spectrometry
7.1 Principles
- Ionization methods (MALDI, ESI)
- Mass analyzers
7.2 Applications
- Protein identification and characterization
- Peptide sequencing
- Analysis of post-translational modifications
8. Cell Culture Techniques
8.1 Basics of Cell Culture
- Types of cell cultures: primary, immortalized, stem cells
- Sterile technique and aseptic handling
8.2 Cell Culture Media
- Components and preparation
- Serum and supplements
8.3 Cell Maintenance
- Passaging and subculturing
- Cell counting and viability assays
- Cryopreservation and thawing
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