HC70AL Spring 2006 Syllabus

Honors Collegium 70AL: Gene Discovery Laboratory Spring 2006

sponsored by Howard Hughes Medical Institute & National Science Foundation

Week One
Date	Topic
4/03/06	Introduction to Seed Development & Research -- Anhthu Bui Data Recording & Organization - Introduction to the Webbook and Lab Blue Books -- Brandon Le
4/04/06	Experiment ONE - Introduction to General Molecular Biology Techniques
	Intro: Lab Orientation and Tour Intro: Introduction to Proper Micropipetting Techniques
	A. Proper Micropipetting Techniques Accuracy/Precision Experiments Gel Electrophoresis of Plasmid DNA Sowing Seeds from Wild Type (Ecotype Col-0) and SALK Lines
4/06/06	Experiment ONE - Introduction to General Molecular Biology Techniques
	Intro: Introduction to Polymerase Chain Reaction (PCR) Intro: Introduction to DNA Sequencing Intro: Introduction to Sizing DNA on Agarose Gel
	B. Polymerase Chain Reaction (PCR) & Sequencing Setting up a Gene-Specific Polymerase Chain Reaction Gel Electrophoresis of Gene-Specific PCR Products Determining DNA Concentration Using Fluorometer Sequencing of Gene-Specific Products
Week Two
Date	Topic
4/10/06	Introduction to Bioinformatics - How to Analyze DNA Sequences -- Brandon Le Introduction to Knockout Screens and Genetics -- Anhthu Bui
4/11/06	Experiment ONE - Introduction to General Molecular Biology Techniques
	B. Polymerase Chain Reaction (PCR) & Sequencing Gel Electrophoresis of Gene-Specific Products
	Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (GENE ONE)
	Intro: Introduction to Genomic DNA Extraction
	A. Extraction of Genomic DNA Set I Tissue Collection from Plants Isolating Genomic DNA from Wild Type and SALK Lines Gel Electrophoresis of Genomic DNA Determining DNA Concentration Using Fluorometer Diluting Genomic DNA
4/13/06	Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (GENE ONE)
	Intro: Review of Genetics and Genotyping -- Mike Gavino
	B. Determination of Genotype Set I Determining Genotype of SALK Plants Using PCR
	C. Extraction of Genomic DNA Set II Tissue Collection from Plants Isolating Genomic DNA from Wild Type and SALK Lines Gel Electrophoresis of Genomic DNA Determining DNA Concentration Using Fluorometer Diluting Genomic DNA
Week Three
Date	Topic
4/17/06	Introduction to Bioinformatics - Annotating Genes -- Brandon Le Review of Webbook and Data Organization -- Brandon Le
4/18/06	Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (Gene ONE)
	Intro: Review of DNA Sequencing -- Mike Gavino
	B. Determination of Genotype Set I Gel Electrophoresis of PCR Product (Section B on 4/13/06)
	D. Determination of T-DNA Insertion Site Set I Discussion of PCR Results Purification of PCR Products Determining DNA Concentration Using Nanodrop Spectrophotometer Sequencing PCR Product with T-DNA and Gene-Specific Primer
	E. Determination of Genotype Set II Determining Genotype of SALK Plants Using PCR
4/20/06	Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (GENE ONE)
	Intro: Introduction to ESTDB and DNA Sequence Analysis -- Brandon Le
	D. Determination of T-DNA Insertion Site Set I Analysis of Sequenced PCR Product (in class)
	E. Determination of Genotype Set II Gel Electrophoresis of PCR Product (Section E on 4/18/06)
Week Four
Date	Topic
4/24/06	Introduction to Gene Expression - RT-PCR and Microarrays -- Brandon Le Team 1 Presents & Discuss Data from Experiment ONE & TWO
4/25/06	Experiment THREE - RNA Isolation and RT-PCR Analysis
	Intro: Introduction to RNA Isolation and Analysis of RNA -- Anhthu Bui
	A. RNA Isolation Preparation & Decontamination of Equipment for RNA Work Isolating Total RNA from Wild type Seeds and Leaves Removal of Genomic DNA from Isolated Total RNA with DNase I Determining RNA Concentration Using Nanodrop Spectrophotometer Gel Electrophoresis of Total RNA (Before and After DNase Treatment)
4/27/06	Experiment THREE - RNA Isolation and RT-PCR Analysis
	Intro: Discussion of Total RNA Quality Intro: Introduction to cDNA Synthesis & RT-PCR
	B. cDNA Synthesis Synthesizing cDNAs from Isolated Total RNA
	C. RT-PCR Amplification of cDNA Using PCR
4/28/06	Experiment THREE - RNA Isolation and RT-PCR Analysis
	C. RT-PCR Gel Electrophoresis of RT-PCR Products
Week Five
Date	Topic
5/01/06	Introduction to Cloning of Promoters -- Tomo Kawashima Team 2 Presents & Discuss Data from Experiment THREE
5/02/06	Experiment FOUR - Amplification & Cloning a Promoter Region
	Intro: Introduction to Amplification & Cloning of Promoters -- Javier Wagmaister Demo: Observation of Promoter::GFP Lines
	A. Amplification of a Promoter Region Amplification of a Promoter Region Using PCR Gel Electrophoresis of PCR Product Ligating PCR Product into a Plasmid Vector pZero-Blunt TOPO
5/04/06	Experiment FOUR - Amplification & Cloning a Promoter Region
	Intro: Introduction to Transformation & Bacterial Techniques -- Anhthu Bui
	B. Transformation of E. coli Cells Transformation of E. coli Competent Cells with Ligation Mixtures Growing Transformed E. coli Cells in SOC Medium Spreading Transformed E. coli Cells on LB + Antibiotic Plates Incubating Cells Overnight at 37oC
5/05/06	Experiment FOUR - Amplification & Cloning a Gene Promoter Region
	B. Transformation of E. coli Cells Counting Blue and White Bacterial Colonies
Week Six
Date	Topic
5/08/06	Characterization of Mutant Plants Using Microscopy -- Anhthu Bui Team 1 Presents & Discuss Data from Experiment FOUR
	Note: Inoculation of LB Broth with Selected Bacterial Colonies
5/09/06	Experiment FOUR - Amplification & Cloning a Promoter Region
	Intro: Introduction to Plasmid DNA Preparation -- Mike Gavino
	C. Isolation & Verification of Recombinant Plasmid DNA Isolating Plasmid DNA from 4 Colonies Determining Plasmid DNA Concentration Verification of Recombinant Plasmid via Restriction Enzyme Analysis Gel Electrophoresis of Restriction Digested Plasmid DNA
5/11/06	Experiment FIVE - Observation & Characterization of Known & Unknown Mutants
	A. Observation of Plant & Seed Phenotype Examine and Compare Wild Type and Mutant plants If Mutant Plant is Homozyogus à Open Silique & Observe Seeds If Mutant Plant is Heterozygous à Open Silique & Count White/Green Seeds
	B. Characterization of Mutant Seeds Using Microscopy Fix Wild Type and Mutant Seeds in Fixative for Nomarski Optics Microscopy Make Appointment to Use Nomarski Optics Microscope (Appointments should be made from 5-15-06 to 5-26-06)
Week Seven
Date	Topic
5/15/06	Ethics in Research -- Professor Goldberg
5/16/06	Experiment FOUR - Amplification & Cloning a Gene Promoter Region
	C. Isolation & Verification of Recombinant Plasmid DNA Sequencing of Recombinant Plasmid DNA
	Experiment SIX - Screening SALK T-DNA Mutagenesis Lines (GENE TWO)
	A. Extraction of Genomic DNA Tissue Collection from Plants Isolating Genomic DNA from Wild Type and SALK Lines Gel Electrophoresis of Genomic DNA
5/18/06	Experiment FOUR - Amplification & Cloning a Gene Promoter Region
	C. Isolation & Verification of Recombinant Plasmid DNA Analyzing and Verifying Promoter DNA Sequence
	Experiment SIX - Screening SALK T-DNA Mutagenesis Lines (GENE TWO)
	A. Extraction of Genomic DNA Determining DNA Concentration Using Fluorometer Diluting Genomic DNA
	B. Determination of Genotype Determining Genotype of SALK Plants Using PCR
Week Eight
Date	Topic
5/22/06	How to Prepare and Present Research Data -- Professor Goldberg -- Discussion of Data from Experiment FIVE
5/23/06	Experiment SIX - Screening SALK T-DNA Mutagenesis Lines (GENE TWO)
	B. Determination of Genotype Gel Electrophoresis of PCR Product (Section B on 5/19/05)
	Experiment SEVEN - RNA Isolation and RT-PCR Analysis
	A. RT-PCR Amplification of cDNA (Isolated Week 4) Using PCR
	Experiment EIGHT - Amplification & Cloning a Gene Promoter Region
	A. Amplification of a Promoter Region Amplification of a Promoter Region Using PCR Gel Electrophoresis of PCR Product Ligating PCR Product into a Plasmid Vector pZero-Blunt TOPO
5/25/06	Experiment SEVEN - RNA Isolation and RT-PCR Analysis
	A. RT-PCR Gel Electrophoresis of RT-PCR Products
	Experiment EIGHT - Amplification & Cloning a Gene Promoter Region
	B. Transformation of E. coli Cells Transformation of E. coli Competent Cells with Ligation Mixtures Growing Transformed E. coli Cells in SOC Medium Spreading Transformed E. coli Cells on LB + Antibiotic Plates Incubating Cells Overnight at 37oC
5/26/06	Experiment EIGHT - Amplification & Cloning a Gene Promoter Region
	B. Transformation of E. coli Cells Counting Blue and White Bacterial Colonies
Week Nine
Date	Topic
5/29/06	Memorial Holiday -- No Class
	Note: Inoculation of LB Broth with Selected Bacterial Colonies
5/30/06	Experiment EIGHT - Amplification & Cloning a Gene Promoter Region
	C. Isolation & Verification of Recombinant Plasmid DNA Isolating Plasmid DNA from 4 Colonies Determining Plasmid DNA Concentration Verification of Recombinant Plasmid via Restriction Enzyme Analysis Gel Electrophoresis of Restriction Digested Plasmid DNA
6/01/06	Experiment EIGHT - Amplification & Cloning a Gene Promoter Region
	C. Isolation & Verification of Recombinant Plasmid DNA Sequencing of Recombinant Plasmid DNA
Week Ten
Date	Topic
6/05/06	Discuss Data From Experiment EIGHT
6/06/06	Experiment EIGHT - Amplification & Cloning a Gene Promoter Region
	C. Isolation & Verification of Recombinant Plasmid DNA Analyzing and Verifying Promoter DNA Sequence
	Preparation for Oral Presentations
	Finish any loose-end experiments Summarize ALL Research Data Organize Blue Books and Webbook
6/08/06	Final Oral Presentations