Honors Collegium 70AL: Gene Discovery Laboratory Spring 2005

sponsored by Howard Hughes Medical Institute & National Science Foundation



Week One
Date Topic
4/04/05 Introduction to Seed Development & Research -- Professor Goldberg
Data Recording & Organization - Introduction to the Webbook and Lab Blue Books -- Brandon Le
4/05/05 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
4/07/05 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
4/11/05 Introduction to Bioinformatics - How to Analyze DNA Sequences -- Brandon Le
Introduction to Knockout Screens and Genetics -- Anhthu Bui
4/12/05 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
4/14/05 Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (GENE ONE)
  Intro: Review of Genetics and Genotyping
  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
4/18/05 Introduction to Bioinformatics - Annotating Genes -- Brandon Le
Review of Webbook and Data Organization --
Brandon Le
4/19/05 Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (Gene ONE)
  Intro: Review of DNA Sequencing
  B. Determination of Genotype Set I
Gel Electrophoresis of PCR Product (Section B on 4/14/05)
  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
4/21/05 Experiment TWO - Screening SALK T-DNA Mutagenesis Lines (GENE ONE)
  Intro: Introduction to ESTDB and DNA Sequence Analysis
  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 4/19/05)
Week Four
Date Topic
4/25/05 Introduction to Gene Expression - RT-PCR and Microarrays -- Brandon Le
Team 1   Presents & Discuss Data from Experiment ONE & TWO
4/26/05 Experiment THREE - RNA Isolation and RT-PCR Analysis
Intro: Introduction to RNA Isolation and Analysis of RNA
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)
4/28/05 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
4/29/05 Experiment THREE - RNA Isolation and RT-PCR Analysis
C. RT-PCR
Gel Electrophoresis of RT-PCR Products
Week Five
Date Topic
5/02/05 Introduction to Cloning of Promoters -- Tomo Kawashima
Team 2 Presents & Discuss Data from Experiment THREE
5/03/05 Experiment FOUR - Amplification & Cloning a Promoter Region
Intro: Introduction to Amplification & Cloning of Promoters
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
5/05/05 Experiment FOUR - Amplification & Cloning a Promoter Region
Intro: Introduction to Transformation & Bacterial Techniques
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
5/06/05 Experiment FOUR - Amplification & Cloning a Gene Promoter Region
B. Transformation of E. coli Cells
Counting Blue and White Bacterial Colonies
Week Six
Date Topic
5/09/05 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
5/10/05 Experiment FOUR - Amplification & Cloning a Promoter Region
Intro: Introduction to Plasmid DNA Preparation
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
5/12/05 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 5-16-05 to 5-27-05)
Week Seven
Date Topic
5/16/05 Ethics in Research -- Professor Goldberg
5/17/05 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
5/19/05 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
5/23/05 Team 2 Presents & Discuss Data from Experiment FIVE
5/24/05 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

5/26/05 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
5/27/05 Experiment EIGHT - Amplification & Cloning a Gene Promoter Region

B. Transformation of E. coli Cells
Counting Blue and White Bacterial Colonies

Week Nine
Date Topic
5/30/05 How to Prepare and Present Research Data -- Professor Goldberg
Team 1 Presents & Discuss Data from Experiments SIX & SEVEN
Note: Inoculation of LB Broth with Selected Bacterial Colonies
5/31/05 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

6/02/05 Experiment EIGHT - Amplification & Cloning a Gene Promoter Region
C. Isolation & Verification of Recombinant Plasmid DNA
Sequencing of Recombinant Plasmid DNA
Week Ten
Date Topic
6/06/05 Team 2 Presents & Discuss Data From Experiment EIGHT
6/07/05 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
6/09/05 Final Oral Presentations