Honors Collegium 70AL: Gene Discovery Laboratory Spring 2011

sponsored by National Science Foundation

Course Information:
  Laboratory: Tuesday & Thursday 2 - 6 PM, Terasaki Life Sciences 4128
  Open Laboratory & Research Consultation: Wednesday & Friday 2 - 6 PM, Terasaki Life Sciences 4128
  Introduction To Laboratory Research: Monday 6 - 8 PM, Terasaki Life Sciences 4100
  Office Hours: Friday 1 - 2 PM, Terasaki Life Sciences 4121
Class Handouts and Powerpoint Presentations:
3-28-11 Syllabus (pdf)
Introduction to Seed Development & Research - Dr. Goldberg (pdf)
Data Recording & Organization - Introduction to the WebBook and Lab Research Notebook - Brandon Le (pdf)
Articles For Discussion:
  •  Jose M. Alonso & Joseph R. Ecker, Moving Forward in Reverse: Genetic Technologies to enable genomic-wide phenomic screens in Arabidopsis, Nature (2006), 7:524-536 (pdf)
  •  Jose M. Alonso, et al., Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana, Science (2003), 301:653-657 (pdf)
  •  Brandon Le, et al., Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors, Proc. Natl. Acad. Sci. (2010), 170:8063-8070 (pdf)
  •  Takashi Kuromori, et al., Phenome Analysis in Plant Species Using Loss-of-Function and Gain-of-Function Mutants, Plant Cell Physiol. (2009), 50: 1215-1231 (pdf)
  •  Ronan C. O’Malley & Joseph R. Ecker, Linking genotype to phenotype using the Arabidopsis unimutant collection, Plant Journal (2010), 61:928-940 (pdf)
  •  On Being a Scientist - A Guide to Responsible Conduct in Research, 3rd Ed., National Academy of Sciences (pdf)
    		•
    3-29-11 Sizing DNA on Agarose Gels - Elaine Chiu (pdf)
    3-31-11 Polymerase Chain Reaction (PCR) - Eden Maloney (pdf)
    Genomic DNA Isolation - Kelli Henry (pdf)
    4-04-11 How to Sequencing DNA - Dr. Goldberg (pdf)
    4-05-11 Knocking-Out Seed Transcription Factor Genes - Dr. Goldberg (pdf)
    4-07-11 Using Bioinformatics to Analyze DNA Sequences - Brandon Le (pdf)
    Plant Genotyping - Eden Maloney and Elaine Chiu (pdf)
    4-11-11 Introduction of Bioinformatics - Brandon Le (pdf)
    4-18-11 RNA and Tools For Studying Differential Gene Expression During Seed Development - Dr. Goldberg (pdf)
    Articles For Discussion:
  •  William A. Haseltine, Discovering Genes for New Medicine, Scientific American, March 1997, 92-97 (pdf)
    		•
    4-25-11 Research Ethics Case Study Discussion - Dr. Goldberg (pdf)
    5-9-11 How To Give A Research Talk - Dr. Goldberg (pdf)
    Lab Schedule:
    Week One
    Date Topic
    3/28/11 Introduction to Seed Development & Research - Professor Bob Goldberg
      Data Recording & Organization - Brandon Le
    3/29/11 Experiment ONE - Introduction to General Molecular Biology Techniques
      Introduction 1: Lab Orientation & Tour - Kelli Henry, Brandon Le, & Jungim Hur
    Introduction 2: Lab Safety - Kelli Henry
    Introduction 3: Proper Micropipetting Techniques - Jungim Hur
    Introduction 4: Sizing DNA on Agrose Gels - Elaine Chiu
      I & II. Proper Micropipetting Techniques
    Accuracy/Precision Experiments
    Gel Electrophoresis of Plasmid DNA
      Experiment FOUR - Screening Salk T-DNA Mutangenesis Line (GENE TWO)
      Introduction 5: Plant Growth Center Tour - Kelli Henry, Brandon Le, & Jungim Hur
      I. Sowing Seeds and Growing Plants
    Sowing Arabidopsis Seed From Wild-Type (Ecotype Col-0) and Salk Lines
    3/31/11 Experiment ONE - Introduction to General Molecular Biology Techniques
      Introduction 1: Polymerase Chain Reaction (PCR) - Eden Maloney
      III. Polymerase Chain Reaction (PCR) & Sequencing PCR Product
    Setting up a Gene-Specific Polymerase Chain Reaction
    Gel Electrophoresis of Gene-Specific Products
      Experiment TWO - Screening Salk T-DNA Mutangenesis Line (GENE ONE)
      Introduction 2: Genomic DNA Isolation - Kelli Henry
      A1. Isolation of Genomic DNA - Set I
    Tissue Collection from plants
    Isolating Genomic DNA from Wild-type and Salk Lines
    Determining DNA Concentration Using Nanodrop Spectrophotometer
    Gel Electrophoresis of Genomic DNA
    Week Two
    Date Topic
    4/04/11 Introduction to Sanger DNA Sequencing - Professor Bob Goldberg
    4/05/11 Experiment ONE - Introduction to General Molecular Biology Techniques
      Introduction 1: Introduction to Knockout Screens and Genetics - Professor Bob Goldberg
      IV & V. Prurifying & Sequencing PCR Product
    Purifying PCR Products
    Determining DNA Concentration Using Nanodrop Spectrophotometer
    Sequencing of Gene-Specific Products
      Experiment TWO - Screening Salk T-DNA Mutangenesis Line (GENE ONE)
      II. Extraction of Genomic DNA - Set I
    Diluting Genomic DNA
      II. Extraction of Genomic DNA - Set II
    Tissue Collection from plants
    Isolating Genomic DNA from Wild-type and Salk Lines
    Determining DNA Concentration Using Nanodrop Spectrophotometer
    Gel Electrophoresis of Genomic DNA
    4/7/11 Experiment ONE - Introduction to General Molecular Biology Techniques
      Introduction 1: Using Bioinformatics to Analyze DNA Sequences - Brandon Le
      VI. Retrieving and Analyzing DNA Sequences
      Experiment TWO - Screening Salk T-DNA Mutangenesis Line (GENE ONE)
      Introduction 2: Plant Genotyping - Eden Maloney and Elaine Chiu
      III. Determination of Genotype of Salk Plants Using PCR
    Determining Genotype of Salk Plants Using PCR
      II. Extraction of Genomic DNA - Set II
    Diluting Genomic DNA
    Week Three
    Date Topic
    4/11/11 Introduction to Bioinformatics - Brandon Le
      Discussion of Data From Experiment ONE - Professor Bob Goldberg
    Experiment ONE Lab Report Due
    4/12/11 Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (GENE ONE)
      Introduction: Review of Plant Genotyping - Eden Maloney and Elaine Chiu
      IV. 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 Salk Primer
      V. Determination of Genotype - Set II
     Determining Genotype of Salk Plants Using PCR
    4/14/11 Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (GENE ONE)
      Introduction: Using Bioinformatics to Analyze DNA Sequences - Brandon Le
      III. Determination of Genotype - Set II
     Gel Electrophoresis of PCR Product (From 4-12-11)
      III. Observation of the Mature Plant Phenotype
      IV. Determination of T-DNA Insertion Site - Set I
     Analysis of Sequenced PCR Product - Brandon Le
    Week Four
    Date Topic
    4/18/11 Introduction to Gene Expression - RT-PCR, Microarray and RNA-Seq - Professor Bob Goldberg
      Discussion of Data From Experiment TWO - Professor Bob Goldberg
    Experiment Two Lab Report Due
    4/19/11 Experiment THREE - Identifying Features of Mutant Seeds Using Nomarski Microscopy (GENE ONE)
      Introduction: Observing Seeds for Mutant Phenotypes - Eden Maloney
    Introduction: Nomarski Microscopy - Kelli Henry
      I. Observation of Seed Phenotypes by Light Microscopy
    Examine and Compare Wild Type and Mutant Seeds
    Fix Seeds for Nomarski Microscopy
    Make Appointment to Use Nomarski Optics Microscopy
    4/21/11 Experiment THREE - Identifying Features of Mutant Seeds Using Nomarski Microscopy (GENE ONE)
      II. Observation of Seed Phenotypes by Nomarski Microscopy
    Week Five
    Date Topic
    4/25/11 Research Ethics Case Study Discussion - Professor Bob Goldberg
    4/26/11 Experiment FOUR- Screening Salk T-DNA Mutagenesis Lines (GENE TWO)
      Introduction: Review of Data Organiztion - Eden Maloney
      II. Extraction of Genomic DNA - Set I
     Tissue Collection from Plants
    Isolation Genomic DNA from Wild Type and Salk Lines
    Determining DNA Concentration Using Nanodrop Spectrophotometer
    Gel Electrophoresis of Genomic DNA
    Diluting Genomic DNA
    4/28/11 Experiment THREE - Identifying Features of Mutant Seeds Using Nomarski Microscopy (GENE ONE)
      Introduction: Chi-Square Analysis - Elaine Chiu
      Experiment FOUR - Screening Salk T-DNA Mutagenesis Lines (GENE TWO)
      III. Determination of Genotype - Set I
     Determining Genotype of Salk Plants Using PCR
    Gel Electrophoresis of PCR Product
    Week Six
    Date Topic
    5/02/11 No Class
    5/03/11 Experiment FOUR - Screening Salk T-DNA Mutangenesis Lines (GENE TWO)
      II. Extraction of Genomic DNA - Set II
     Tissue Collection from Plants
    Isolation Genomic DNA from Wild Type and Salk Lines
    Determining DNA Concentration Using Nanodrop Spectrophotometer
    Gel Electrophoresis of Genomic DNA
    Diluting Genomic DNA
    5/05/11 Experiment FOUR - Screening Salk T-DNA Mutangenesis Lines (GENE TWO)
    III. Determination of Genotype - Set II
    Determining Genotype of Salk Plants Using PCR
    Gel Electrophoresis of PCR Product
    Week Seven
    Date Topic
    5/09/11 How to Prepare and Present Research Data - Professor Bob Goldberg
      Discussion of Data From Experiment FOUR - Professor Bob Goldberg
    Experiment Three Lab Report Due
    5/10/111 Experiment FOUR - Screening Salk T-DNA Mutangenesis Lines (GENE TWO)
    C. Determination of T-DNA Insertion Site
    Purification of PCR Products
    Determining DNA Concentration Using Nanodrop Spectrophotometer
    Sequencing PCR Product with T-DNA and Gene-Specific Salk Primer
    D. Observation of the Mutant Plant Phenotype
    5/12/11 Experiment FOUR - Screening Salk T-DNA Mutangenesis Lines (GENE TWO)
    C. Determination of T-DNA Insertion Site
    Analysis of Sequenced PCR Product
    Gene Annotation Worksheet for Gene 2
      Experiment FIVE - Identifying Features of Mutant Seeds Using Nomarski Microscopy (GENE TWO)
    I. Observation of Seed Phenotypes by Light Microscropy
    Examine and Compare Wild Type and Mutant Plants
    Fix Seeds for Nomarski Microscopy
    Make Appointment to Use Nomarski Optics Microscopy
      Experiment SIX - Amplifying & Cloning a Gene Upstream Region (GENE TWO)
      Introduction: Amplification & Cloning of Upstream Regions - Kelli Henry
    A. Amplification of a Promoter Region
    Amplification of a Promoter Region Using PCR
    Gel Electrophoresis of PCR Product
    Week Eight
    Date Topic
    5/16/11 Introduction to Cloning of Promoters - Kelli Henry
      Discussion of Data from Experiment FOUR
    Experiment Four Lab Report Due
    5/17/11 Experiment FIVE - Identifying Features of Mutant Seeds Using Nomarski Microscopy (GENE TWO)
    II. Observation of Seed Phenotypes by Nomarski Microscropy
      Experiment SIX - Amplifying & Cloning a Gene Upstream Region (GENE TWO)
      Introduction: Transformation & Bacterial Techniques - Kelli Henry
    Demonstration: Observation of Promoter::GUS-GFP Lines - Kelli Henry
    A. Amplification of a Promoter Region
     Ligating PCR Product into a Plasmid (pENTR/D-TOPO Vector)
    B. Transformation of E. Coli Cells
    Transformation of E. Coli Competent Cells with Ligation Mixtures
    Incubating Cells Overnight at 37 °C
    5/19/11 Experiment SIX - Amplifying & Cloning a Gene Upstream Region (GENE TWO)
    C. Isolation & Verification of Recombinant Plasmid DNA
    Isolating Plasmid DNA from Four Colonies
    Determining Plasmid DNA Concentration Using Nanodrop Spectrophotometer
    Verification of Recombinant Plasmid via Restriction Enzyme Analysis
    Gel Electrophoresis of Restriction Digested Plasmid DNA
    Week Nine
    Date Topic
    5/23/11 Discussion - What Did I Learn in HC70A and HC70AL? - Professor Bob Goldberg
      Experiment Five Lab Reports Due
    Discussion of Data From Experiment FIVE - Professor Bob Goldberg
    5/24/11 Experiment SIX - Amplifying & Cloning a Gene Upstream Region (GENE TWO)
    C. Isolation & Verification of Recombination Plasmid DNA
    Sequencing Recombinant Plasmid DNA
    General Laboratory
    Finish Experiments, Summarize Data & Prepare PowerPoint Presentation
    5/26/11 Experiment SIX - Amplifying & Cloning a Gene Upstream Region (GENE TWO)
    C. Isolation & Verification of Recombination Plasmid DNA
    Analyzing and Verifying Promoter DNA Sequence
    General Laboratory
    Finish Experiments, Summarize Data & Prepare PowerPoint Presentation
    Week Ten
    Date Topic
    5/30/11 Memorial Day - No Class
    5/31/11 Discussion of Data From All Experiments - Professor Bob Goldberg
    Experiment Six Lab Report Due
    Clean-up Benches, Summarize Data, & Organize Lab Notebook & Webbook
    Organize & Practice Research Talks
    6/01/11 Exit Interviews With Professor Bob Goldberg
    6/02/11 All-Class Research Symposium
    11th Week Exit Interviews With Professor Bob Goldberg