TECHNIQUES IN BIOTECHNOLOGY (BIO
461)
Spring 2010
Tentative Course Syllabus
Class
Meets MW 3:30 to
6:00 in FSC 132
Course
Instructors: Drs.
William Patrie and Lucinda Elliott
Offices:
Dr. Patrie (FSC
152), Dr. Elliott (FSC 153)
Campus
Phones: Dr. Patrie
(477-1400), Dr. Elliott (477-1504)
Email
Addresses: Dr.
Patrie (wjpatr@ship.edu), Dr. Elliott (lhelli@ship.edu)
Home
Pages: Dr. Patrie (www.ship.edu/~wjpatr/), Dr. Elliott (www.ship.edu/~lhelli/)
Office
Hours: Dr.
Patrie, MWF 10-11; MTW 1-2
Dr. Elliott, M/W 11:00-12:00,
Tue 9:00-11:00; Fri 1:00-3:00
|
Date |
Laboratory
Exercise |
|
|
Jan Wed |
20 |
Overview
of the course and pre-assessment quiz Introduction/
Background/ Lab Safety Assignment: Download MSDS forms, provide
summary/ assessment; solution problems Immunology:
Overview and Review Lab #1: Tissue Culture Techniques
(Non-adherent Hybridoma cell line) |
|
Mon |
25 |
Lab #2: Care and handling on of mice;
Harvesting of lymphoid organs: Preparation of single cell suspensions Assignment: Dilutions and Cell Counts |
|
Wed |
27 |
Immunology
Lecture: Cytokines |
|
Feb Mon |
01 |
Lab #3: In vitro stimulation of spleen
cells Collection
of day 5 secondary antiserum from mice Lab Notebook Check |
|
Tue |
02 |
Collection
of culture supernatant and spleen cell lysates to
be stored at -80C |
|
Wed |
03 |
Lab #4: Detection of TNF-a expression by RT-PCR; Isolation
of total RNA from spleen cells and preparation of cDNA |
|
Mon |
08 |
Lab #3 (cont): Collection of day 12 secondary
anti-SRBC antiserum from mice Lab #4 (cont): PCR amplification of TNF-alpha cDNA Lab #5: Determination of anti-SRBC titer
by hemagglutination: Set up assay |
|
Wed |
10 |
Lab #4 (cont): Gel analysis of PCR products Lab #5 (cont): Determination of anti-SRBC titer
by hemagglutination: Determine titer |
|
Mon |
15 |
Lab #6: Overview of TNFa ELISA: Procedure and Dilutions Lab #7: Affinity column chromatography of hybridoma monoclonal antibody Lab Notebook Check |
|
Wed |
17 |
Lab #6 (cont): ELISA detection of TNF-alpha
protein (Start 4-5 hrs before lab) Lab #7 (cont): Collection of dialyzed mAb Overview
of independent research project Assignment: Research Proposal |
|
Mon |
22 |
Lab #7 (cont): Determination of monoclonal
antibody concentration by BCA Lecture:
Molecular Biology Overview Lab Notebook Check |
|
Wed |
24 |
Lab #8: Analysis of murine
gamma globulin by size exclusion FPLC Lecture:
DNA forensics |
|
March Mon |
01 |
Lab #9: Forensics DNA Lab: DNA isolation and PCR |
|
Wed |
03 |
Lab #9: Forensics DNA Lab continued Lab Notebook Check Assignment: Journal Article Graduate Literature
Review Due |
|
|
|
Spring Break March 5 (4:00pm) to
March 15 (8:00am) |
|
Mon |
15 |
Research Proposal Due: Begin Independent
Research |
|
Wed |
17 |
Journal Article presentation (Dr. Elliott & Dr. Patrie)
& quiz |
|
Mon |
22 |
Research project |
|
Wed |
24 |
Journal Article presentation (Group 1) & quiz |
|
Mon |
29 |
Research project Written Research Progress Report due |
|
Wed |
31 |
Journal Article presentation (Group 2) & quiz |
|
April Mon |
05 |
Research project Lab Meeting & Research Progress Report (ppt) |
|
Wed |
07 |
Journal Article presentation (Group 3) & quiz |
|
Mon |
12 |
Research Project Written Research Progress Report due |
|
Wed |
14 |
Research project |
|
Mon |
19 |
Research project Lab Meeting & Research Progress Report (ppt) |
|
Wed |
21 |
Research project |
|
Mon |
26 |
Written Research Progress Report due |
|
Wed |
28 |
Post Assessment Quiz Research laboratory journals due |
|
|
|
Final Research Presentations |
Course Design
The course is designed introduce you to the joys and pitfalls of laboratory research. The course is also designed to function as the laboratory components of Immunology and Molecular Biology. During the first half of the course you will be introduced to a number of techniques commonly used in immunology laboratories. Many immunology and biomedical research laboratories use animal models to investigate and elucidate physiological, cellular and molecular mechanisms in normal and diseased hosts. Rodents are particularly popular because they are easy to breed and handle as well as cheap to house. The use of primary and malignant cell tissue culture lines is also commonly used to elucidate cell and molecular mechanisms. Because many of you are planning careers in basic and biomedical research, we believe that hands on experience with animals and tissue culture will make your more competitive when you choose to enter graduate school and/or the job market. In this course you will be instructed on the use and handling of mice in the laboratory. You will immunize these mice with an antigen (Sheep Red Blood Cells) and monitor the immune response in harvested lymphoid organs using both immunologic and molecular techniques. You will also have the opportunity to increase your tissue culture skills. In the second half of the course you will use molecular biology techniques to clone and characterize a gene that is expressed during the immune response in mouse lymphoid tissue harvested from the immunized mice.
Each
student will be expected to keep a detailed laboratory notebook, which
should contain the materials & methods, results, conclusions and discussion
for each exercise. The laboratory notebook is your journal for the course and
thus the more information you include the better. The instructors will
check (& grade) the lab notebooks several times during the first half of
the semester to make sure they are up to date and complete. The lab manual, which contains background
information and detailed protocols for the immunodiagnostics portion of this course is posted on blackboard. It is suggested that you copy the lab manual
to a folder on your computer. Before you
come to lab read through the background information and copy the title, purpose
and detailed procedure into your lab journal.
Do not come to lab unprepared! We reserve the right to give pop quizzes in lab
if we believe the class as a whole is unprepared.
Students will work in teams of three or four to complete an independent research project. Each student will be responsible for maintaining their own laboratory journal for the shared project. Independent projects will include weekly written progress reports, which summarize the experimental procedures and results obtained. The report should also include all problems encountered in the project and how these problems were solved. Details of what each group member contributed to the project must also be included in the progress report. Lab meetings will be held every two weeks to present progress reports in the form of a ppt to the rest of the class. The purpose of these meetings is to generate discussion among the class and give you feedback that may help you with your project.
The research project for the course will involve primer design for amplification & cloning of the cDNA of a gene of interest expressed in activated murine spleen cells. The cloned products will be characterized by restriction mapping and DNA sequencing. The lab schedule is a guideline that may vary with each individual, particularly toward the end of the semester. Successful completion of the project will require a group effort and some time spent outside the designated class time. Group members will be required to grade each other for individual contribution to the project.
Grading
Pre-and
post assessment exams will be administered to determine your progress in the
course. The pre-assessment quiz will not
count in the final grade, but the post-assessment quiz will. Individual
laboratory journals covering lab exercises 1 through 9 will be graded
periodically throughout the first half of the semester. Individual laboratory journals of the
research project will due on the last day of class so that they can be graded
and returned during the final. Research proposals and progress reports (written
and ppt presentations) will be graded for each
group. In addition, each group will be
responsible for choosing and presenting a recent article from a major peer
reviewed journal that is related to the gene of interest for their research
project. The group will also prepare a 15 pt quiz on the article that
will be administered to the remaining members of the class. The instructors will present the first
article to demonstrate what is expected. Lab assignments and problem sets
on the principles of techniques covered in the course will be scattered
throughout the semester and will be graded on an individual basis.
Graduate students will be expected to complete a 10 page literature review of
the gene / gene product of interest for their group. The review must be formatted with 1.5 spaces
between lines and review a minimum of 10 primary research articles which are
referenced numerically and cited throughout the paper.
The final grade will be based on a total of 750points (800 for graduate students) and will include:
Lab Notebook checks @ 20pts each 100
Lab Notebook for Independent Project 100
Progress Reports @ 20pts each 100
Assignments and Problem Sets: 100
Research Proposal 100
Journal Club Quizzes @ 15pts each 60
Individual contribution/independence 40 (Graded by peers)
Final Presentation of Research project 100
Post Assessment Exam 50
Total 750
Graduate Literature Review 50
Your final letter grade will be computed according to the following scale:
|
90-100% of total
points = A |
70-76% = C |
Grades will be posted on
blackboard
Any student who requires special accommodations for taking tests or notes, should make an appointment to see Dr. Patrie or Dr. Elliott in order to make appropriate arrangements.
Grading Rubric for Lab Journal
Checks (20pts each)
1.
Table of Contents with dates and page numbers (5pts)
2.
Individual Lab Exercises: Note: Right and Left sides of the manual (15pts)
a.
Title, date and
purpose of the exercise: Right side
b.
Reagents (Buffer formulations, Catalog # and
Sources, Concentrations, Dilutions etc): Left side
c.
Bulleted detailed
procedure
including steps, amounts of reagents added, incubations times, relevant
schematics of how plates or tubes were prepared, PCR program (if relevant): Right
side
d.
Results: Calculations, figures and tables
(with appropriate figure legends and descriptive titles. Left side
e.
Conclusions and
Discussion: Narrative
paragraph summarizing the conclusions and discussion of the results. Right
Side
Grading Rubric for Research Project
Lab Journal (100pts)
1. Table of Contents
(5pts): Are the dates and page numbers included
2. Experimental Design
(25pt): Content Does each experiment have the following
a. Purpose
b. Detailed Procedure
(with buffers, dilutions, and PCR program details)
c. Calculations
d. Results (Including
figures and graphs with figure legends)
e. Conclusions
f.
Discussion
3. Content & Clarity
(15pts): Are the experiments clearly
written and easy to understand. Did you
leave out important details? Are the figures
clearly labeled? Did you include the
following information
a. Blast sequence info on
gene of interest
b. Design of primers
c. Restriction Maps and
Determination of fragment sizes based on orientation
d. Sequence data and
blast information on your cloned cDNA.
4. Organization
(5pts): Are the experimental protocols
and results in logical order. Can we
match the results, figures and discussions with the experiments?
Grading Rubric for Journal Club and
Research Presentations
|
|
Possible pts |
Score |
|
PowerPoint:
Organization and Visual Effects |
15 |
|
|
Background
Information and Introduction |
15 |
|
|
Understanding
& explanation of experimental design |
20 |
|
|
Understanding
& explanation of results |
20 |
|
|
Understanding
& explanation of conclusions |
15 |
|
|
Ability
to answer questions |
15 |
|