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Document Outline
Syllabus
Course objectives:
Course logistics:
Grading:
Course Grading Scale:
Description of Course Grading Items:
Student Responsibilities:
Policies:
Basic Molecular Lab Techniques
Solutions and dilutions in molecular biology:
Definitions:
Dilutions:
Use of the pipet
Lab temperatures:
Pipetting exercise:
Sterile Technique
Sterile technique exercise:
Growth of E. coli
Glossary of E. coli cloning terms
How to streak bacteria on LB plates
Gene Structure
What is a gene?
Genetics and C. elegans
Introduction
Growth and maintenance
Sexual forms
Life Cycle
Genetics
Why use C. elegans?
Brief history of C. elegans research and key discoveries
Reading Scientific Manuscripts
How to read and understand a scientific article
Step-by-Step Instructions for Reading a Primary Research Article
Purification of DNA
Genomic DNA extraction using a kit (GeneJet Genomic Purification Kit)
Genetic basis of sensation I
Chemotaxis.
Plasmid miniprep
Plasmid miniprep protocol
Genetic basis of sensation II
Mechanosensation.
Gel electrophoresis
Genetic basis of disease I
Cancer.
Parkinson’s disease.
Forward Genetic Screen I
Isolation of mutants
Cultivation of mutants
Induction of male production by heat shock
Worm PCR
Forward Genetic Screen II
Confirmation of mutant phenotype in F2
Cryopreservation of mutant strains.
Freezing Worms with Trehalose-DMSO
Backcrossing of mutants
Complementation
Introduction
A simple test for assigning a mutation to a genetic locus
Conclusion
Mapping of mutation locus.
A multiply fluorescent strain for outcrossing and linkage mapping
Forward Genetic Screen III
Confirmation of complementation/mapping/backcrossing.
Spectrophotometry
Electronic constructs
Introduction
Primer design
Considerations in Primer Design
Specificity:
Melting temperature (Tm)
Primer length:
Product size:
Primer dimers:
Hairpins:
G/C content:
G/C clamp:
Primer design:
Programs for organizing DNA sequences:
in silico cloning:
Electronic Restriction Digestion:
Cryopreservation Verification:
Polymerase chain reaction (PCR)
Introduction
Reaction components:
Guidelines for designing primers:
The typical program:
Setting up the PCR
Troubleshooting
PCR protocol:
Typical PCR components:
Identification of isolated mutants through PCR
PCR fusion
Promoter fusion with GFP
DNA Cloning I
Introduction
Ligation-mediated cloning
Cloning tools
Blunt-end cloning
Preparing the insert:
Preparing the vector backbone:
Ligation with inserts amplified using 5’-phosphorylated primers (not done in class):
Ligation with inserts amplified using non-phosphorylated primers:
Fast Transformation of Mix & Go Competent Cells*
Notes for high efficiency transformation
1. E. coli Strains
2. Incubation Time
3. Prewarming Culture Plates
4. Addition of SOC Medium to Transformation Mixtures (Outgrowth)
5. Culture Conditions
Appendix
SOB Recipe (1 Liter):
SOC Recipe: (100 ml):
Colony PCR protocol:
Cellular manipulations I
Optogenetic ablations with KillerRed.
Cellular manipulations II
Neuronal activation with Channelrhodopsin. (Adapted from Fang-Yeng et al., doi: 10.1098/rstb.2014.0212)
Measuring cell activity with GCaMP.
Restriction digestion of DNA
Restriction enzymes:
Plasmid digestion:
Gel purification with glass wool.
Materials
Results and Discusion
DNA cleanup
Restriction cloning (Subcloning)
Choosing your enzymes
Experimental flowchart
Digestion
Isolate your insert and vector by gel purification:
Ligate your insert into your vector:
RNA interference
Introduction
Use of RNAi in C. elegans
APPENDIX 1:
Agreement to Adhere to Department of Biological Sciences Safety Policies
Safe Lab Practices
APPENDIX 2:
Sample prelabs
APPENDIX 3:
Lab book Scoring Rubric.
APPENDIX 4:
Power Point Pointers
APPENDIX 5:
Filming freely moving worms
Creation of the filming arena
Transferring worms to the filming plate
Filming worms with Micro-Manager
Filming worms with Micro-Manager
Tracking freely moving animals
Summary
General considerations
1. Crawling analysis
Loading movies into ImageJ.
Background subtraction by rolling ball method
Converting movie to binary
Tracking of crawling worms
Summary of analysis
Histogram analysis
2. Swimming analysis – Quantification of body-bends
Removing flickering from 60Hz light-sources
Generating maximum intensity image for background subtraction.
Subtracting maximum projection from movie
Quantification of body bends in movie
NOTES:
APPENDIX 6
Basic Statistic Concepts
APPENDIX 7
Lab Feedback form
APPENDIX 8
Building your own tracking system
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BSC220 Fall 2019
Cell & Molecular Genetics
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