Nguồn ngoài

Investigations in Molecular Cell Biology

The course introduces students to molecular cell biology within the context of a semester-long research project in functional genomics. In the Pathways over Time project, students study the evolutionary conservation of genes in methionine synthesis. Each chapter includes both theoretical background material as well as detailed experimental procedures. Chapters can be used alone or in combination, depending on the course.

Cell and Molecular Biology Biology

Tác giả: Clare M. O’Connor
Nhà xuất bản: Boston College
Nguồn: LibreTexts
Giấy phép: CC BY-NC-SA
Ngôn ngữ: EN
Danh mục: Khoa học cơ bản

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Mục lục· 129 mục

01: Introduction
1.01: Course Design and Learning Goals
1.02: Pathways over Time- our research project
1.03: Course overview
1.04: Acknowledgments
1.05: References
02: Mastering the micropipette
2.01: Using micropipettes correctly
2.02: Using the spectrophotometer to evaluate your pipetting skills
2.03: Light spectroscopy
2.04: Using the GeneSys 20 spectrophotometer
2.05: Exercise 1 - Getting the feel of micropipettes
2.06: Exercise 2 - How NOT to pipette
2.07: Exercise 3 - How precise and accurate are your transfers
2.08: Test yourself
03: Meet the yeast
3.01: Observing microorganisms with light microscopy
3.02: Two very different yeast
3.03: Escherichia coli is a small prokaryotic cell
3.04: Exercise 1 - using the compound light microscope
3.05: Exercise 2 - observing yeast and bacterial cultures
3.06: References
3.07: References
04: Working with Yeast
4.01: Sterile technique
4.02: Yeast growth media
4.03: Yeast growth phases
4.04: Exercise 1 - Streak plates
4.05: Exercise 2 - Spot plates
4.06: Exercise 3 - Estimating cell densities with a spectrophotometer
4.07: Discussion questions
4.08: References
05: Introduction to databases
5.01: Databases organize information
5.02: From the research bench to the database
5.03: Saccharomyces genome project provided the reference sequence
5.04: Exercise 1- Finding gene records in NCBI databases
5.05: Exercise 2- Using the Saccharomyces Genome Database
5.06: Test yourself
5.07: References
06: Analysis of mutant strains
6.01: Cells require sulfur-containing amino acids
6.02: Genetic nomenclature
6.03: Auxotrophs and selective media
6.04: Genetic analyses of methionine biosynthesis
6.05: Biochemistry of the sulfur amino acids
6.06: Exercise 1 - Predicting growth properties of mutant strains
6.07: Exercise 2 - Identifying strains by nutritional requirements
6.08: References
07: Yeast colony PCR
7.01: Polymerase chain reaction overview
7.02: Saccharomyces Genome Deletion Project
7.03: Exercise 1 - Physical properties of PCR primers
7.04: Exercise 2 - Predict the sizes of the PCR products
7.05: Exercise 3 - Design yeast colony PCR
7.06: Exercise 4 - Yeast colony PCR
7.07: Test yourself
7.08: References
08: Agarose gel electrophoresis
8.01: Background
8.02: Prepare the agarose gel
8.03: Sample preparation
8.04: Load and run the agarose gel
8.05: Stain and analyze the agarose gel
8.06: Test yourself
09: Protein Conservation
9.01: References
9.02: Amino acid R groups have distinct chemistries
9.03: Exercise 1 - The 1-letter code for amino acids
9.04: BLAST algorithms are used to search databases
9.05: BLOSUM62 scoring matrix for amino acid substitutions
9.06: Exercise 2 - The BLOSUM62 matrix
9.07: The BLASTP algorithm
9.08: Exercise 3 - Using BLASTP
9.09: Exercise 4 - Multiple sequence alignments
9.10: Investigate S. pombe homologs using Pombase
10: Plasmids
10.01: Plasmids are composed of functional elements
10.02: Plasmid nomenclature
10.03: Plasmids are easily isolated from bacterial cells
10.04: Exercise 1 - Plasmid isolation with the ZyppyTM kit
10.05: References
11: Restriction mapping
11.01: Restriction endonucleases
11.02: DNA molecules have unique restriction maps
11.03: Exercise 1 - Plan the restriction digest
11.04: Exercise 2 - Set up the restriction digests
11.05: Exercise 3 - Analyze the restriction digests on agarose gels
11.06: Test yourself
12: Yeast Transformation
12.01: Transformation alters the phenotype of a cell
12.02: Complementation is often used to isolate transformants
12.03: Experimental considerations
12.04: Replica plates accelerate the screening process
12.05: Exercise 1 - Yeast transformation
12.06: Exercise 2 - Replica plating and complementation
12.07: Test yourself
12.08: References
13: Protein overexpression
13.01: Regulation of the GAL1 promoter
13.02: Preparing protein extracts from yeast cells
13.03: Exercise 1 - Prepare cell cultures for extraction
13.04: Exercise 2 - Preparing cell extracts
13.05: References
13.06: Section 6-
14: SDS-PAGE
14.01: Background
14.02: Casting SDS-PAGE gels
14.03: Running SDS-PAGE gels
14.04: Staining SDS-PAGE gels
14.05: References
15: Western blots
15.01: Antibodies are produced in response to antigens
15.02: Antibodies are produced by lymphocytes
15.03: Plasmid-encoded proteins have C-terminal tags
15.04: Western blots involve many steps
15.05: Exercise 1 - Preparing the membrane replica
15.06: Exercise 2 - Immunodetection
15.07: References
16: Write It Up
16.01: Micro-reports - general guidelines
16.02: Micro-report 1- Selective plating experiment
16.03: Micro-report 2- Yeast colony PCR
16.04: Micro-report 3- Restriction mapping
16.05: Micro-report 4- Complementation analysis
16.06: Micro-report 5- SDS-PAGE and western blot analysis
16.07: Final lab report
16.08: Poster
16.09: References