MCB 473/573

Recombinant DNA Methods and Applications
   
Schedule
Fall 2005
Week 1 Week 5 Week 9 Week 13
Week 2 Week 6 Week 10 Week 14
Week 3 Week 7 Week 11 Week 15
Week 4 Week 8 Week 12 Week 16
Date/Day
Lecture/Lab Focus
Reading/Assignments

Week 1

08/22 M

Project 1: Basic Lab Skills and Lab Preparation

Lecture: Introduction to the course, gene manipulation, cutting and joining of DNA molecules, basic lab math, how to write a prelab

Lab Exercises:

Basic lab math, units of measurements, pipetting exercise, calculations for solution making

Discussion Session: TA - Basic lab math, other, how to keep a lab notebook

None
08/24 W

Project 2: Plasmid DNA Isolation

Lecture: Isolation of nucleic acids, plasmid isolation

Lab Exercises: Preparation for the isolation of plasmid DNA

Exercises: A, B, C

Discussion Session: TA

DUE:
Project 2 Prelab

Project 2 Appendices
Text Section 2.7

Week 2

08/29 M

Lecture: Anatomy of a plasmid vector, other vectors, gene cloning strategies

Exercises: D

Discussion Session: TA

DUE:
Text Chapter 3

08/31 W

Lab Exercises: E, F, G, H, I

Discussion Session: TA - Restriction Mapping, other

DUE:
Text Section 2.1-2.3, 2.5

Week 3

09/05 M
Labor Day - NO CLASSES
None
09/07 W

Lecture: Restriction Enzymes, modifying enzymes, modifying and repairing DNA ends, electrophoresis

MCB Online Class Access demo.

Lab Exercises: Troubleshooting Experiments

Run supplemental experiment

How to analyze gel electrophoresis data

Discussion Session: TA - Restriction Mapping, How to write a lab report

DUE:

Text Chapter 4, 5.1-5.2

Week 4

09/12 M

Project 3: Subcloning Plasmid DNA

Lecture: Model organisms, sub-cloning strategies, transformation, selection, screening

Lab Exercises: Shot gun cloning

A, B

Discussion Session: Troubleshooting, effects of different concentration of vector DNA and insert DNA on ligation, questions for the lab reports

QUIZ #1

DUE:
Project 3 Prelab

Reading Assignment: Cohen et. al. paper for discussion on 9/26
09/14 W

Lecture: Libraries, Introduction to PCR

Lab Exercises: Competent cells and ligation analysis

C, D

Discussion Session: Troubleshooting, log phase cells, transformation, PCR and the PCR screen

DUE:

5.3-5.6

Week 5

09/19 M

Lecture: Screening recombinant clones using PCR

Lab Exercises: Transformation and lab prep

E, F

Discussion Session: Troubleshooting the transformation

DUE:
Project 2 Lab Reports

09/20 T

NOT YOUR REGULARLY SCHEDULED DAY

  • Required Attendance - short exercise

Lab Exercise: Screening

G
NONE
09/21 W

Lecture: Restriction enzyme analysis, DNA Sequencing

Lab Exercise: PCR screen gel assay and Plasmid isolation

H, I

Discussion Session: Troubleshooting, PCR screen, factors that affect PCR

DUE:

Text Section 9.4-9.5

Note:

Prepare your presentation of the assigned section in Cohen et. al. paper

Week 6

09/26 M

Lab Exercise: Digest Gel Assay and Project Data Analysis

J

Discussion Session: Troubleshooting the project experiments and preparation for Lab Meeting presentations.

  • Discuss the Cohen et. al. paper

DUE:
Cohen Paper
09/28 W

Project 4: DNA Sequencing and Introduction to Bioinformatics

Lecture: Introduction to Bioinformatics, NCBI tour: (Elizabeth Kline - UA Library Services)

Lab Exercise: DNA sequencing, and computer exercises

A, computer exercise handout

No Project 4 Prelab (Project 3 and 4 will be integrated)

AB pp 7-28

2nd Half of the class will be in the library computer lab (Rm Information Commons Rm 112A in the main library)

Week 7

10/03 M

Lab Exercise: Purify DNA sequencing reactions and get them ready for drying

A, computer exercise handout

Ex4.A

C, Tour of the GATC Sequencing Facility LSS203

Guest Speaker: Ryan Sprissler

QUIZ #2

SECTION 1

Meet in Koffler 541

We will then go to LSS203 for the tour then to the BLC for the remainder of the class

SECTION 2

Meet in LSS 2nd Floor Lobby

We will then go to LSS203 for the tour then, to the lab to work on the sequencing reactions before we go over to the BLC for the remainder of the class

DUE:

Text Section 9.6-9.9
10/05 W

Lab Exercise: Analyze sequences

B, computer exercise handout

Ex4.B

Class in BLC

Week 8

10/10 M

Exercises: Sequence analysis

Discussion Session: Data Analysis

Class in BLC
10/12 W

LAB Meeting: 1,2,3,4

Lab presentations

DUE:
Project Presentations

LAB MEETING HELD IN LSS340

Week 9

10/17 M

MIDTERM EXAM

Projects 1,2,3,4

2 HRS

10/19 W

Project 5: Isolation and Characterization of 5' Ends of Genes

Lecture: Experiment design

Lab Exercises: Experiment design

Discussion Session: Experiment design, Project 5 Prelab writeup

DUE:

5' RACE System Manual pp 6-17

Topo TA Cloning System Manual pp 1-6, 10-23

VERSAGENE RNA Purification System Manual

PURESCRIPT RNA Purification Manual

TempliPhi Amplification Manual pp 5-12

T4 DNA Ligase

NEB Klenow (Large Fragment Info)

PfxUltima Manual

Project 5 Prelab DUE FRI 10/21 Online Submission

Week 10

10/24 M

Lecture: Isolation of RNA, 5' RACE, Modifying protocols

Lab Exercises: RNA Isolation, electrophoretic assay of RNA, fluorometric assay of RNA

Discussion Session: Experiment progress, plans for next exercise

Combined Project 3 and Project 4 Lab Reports is due

Re-read Text 4.7-4.9

Text 5.1-5.2
10/26 W

Lab Exercises: Experiment plan [continued]

Discussion Session: Experiment progress, plans for next exercise

Lab Progress Report

Text 9.6-9.9

Week 11

10/31 M

Lab Exercises: Experiment plan [continued]

  • RACE, 1st strand synthesis, purification, TdT tailing, ON PCR of dC-tailed cDNA
  • Prep for cloning, fluormetric assay of pCR2.1, Restriction digests of pCR2.1

Discussion Session: Experiment progress, plans for next exercise

Text Chapter 10

How to keep lab note books (from MIT)

Lab Progress Report
11/02 W

Lab Exercises: Experiment plan [continued]

  • Nested PCR if necessary, gels, repair of DNA ends, ligations
  • Prep for cloning cont.
  • Transformations

Discussion Session: Experiment progress, plans for next exercise

  • FRIDAY OPTIONS

Lab Progress Report

Aharoni A, et al Plant Cell. 2004 Nov;16(11):3110-31 to be discussed 11/09 (Obtain the article from PubMed)

Week 12

11/07 M

Lab Exercises: Experiment plan [continued]

  • Clone screening
  • Prep to submit samples for DNA sequencing

Discussion Session: Experiment progress, plans for next exercise
Lab Progress Report
11/09 W

Lab Exercises: Experiment plan [continued]

  • PCR cleanup, plasmid assays, restriction digests, gel assays, flurometric assays
  • Prep to submit samples for DNA sequencing

Discussion Session: PRESENTATION of Aharoni et., al. paper (1 HOUR)

  • FRIDAY OPTIONS

Lab Progress Report

DUE: Aharoni Paper

Week 13

11/14 M

Lab Exercises: Bioinformatics, Experiment plan

Lab Progress Report

11/16 W

Lab Exercises: Bioinformatics, Experiment plan [Wrap up]

  • FRIDAY OPTIONS

 

Week 14

(Thanksgiving Week)

11/21 M

 

Lab Meeting: Project 5

Group Presentations (Individual Presentation Graded)

Grad Student Presentations

QUIZ #3

DUE:
GROUP PRESENTATIONS HELD IN LSS340

TURN IN LAB NOTEBOOKS

11/23 W

Happy Thanksgiving

NO CLASS

Week 15

11/28 M

Project 6: Introduction to Proteomics

Lectures: Expression vectors and the experiment. Guest Speaker: Will Sewell

Tools for Protein Analysis. Guest Speaker: Mike Galligan

Lab Exercises: Protein purification

Text 8.1-8.5

DUE: Project 6 Prelab

 
11/30 W

Lecture: Mass Spectroscopy and Proteomics

Lab Exercises: Run purified protein in SDS-PAGE, analyze results

Discussion Session: Data analysis

DUE: Project 5 Lab Reports

Week 16

12/05 M

Tour of the Proteomics Facility
Core, SWEHSC

Guest Speaker: George Tsaprailis

Meet at the courtyard outside PHARMACY

Section 1, 12:00-1:00

Section 2, 5:00-6:00

  • Quiz#4
  • Exam Review

QUIZ #4
12/07 W

Last day of classes and laboratory sessions

SECTION 1 AND SECTION 2 FINALS

DUE:
Project 6 Lab Report

FINAL EXAM

Koffler541

Return to MCB 473/573 Home