TECHNIQUES IN BIOTECHNOLOGY (BIO 461) Spring 2013

Transcription

1 TECHNIQUES IN BIOTECHNOLOGY (BIO 461) Spring 2013 Class Meets MW 3:30 to 5:50 in FSC 132 Course Instructors: Drs. Lucinda Elliott and William Patrie Offices: Dr. Elliott (FSC 153), Dr. Patrie (FSC 152) Campus Phones: Dr. Elliott ( ), Dr. Patrie ( ) Addresses: Dr. Patrie Dr. Elliott Home Pages: Dr. Patrie ( Dr. Elliott Office Hours: MWF 9:00-10:00; T/R 1:00-2:00 Dr. Patrie Office Hours: MWF 11:00-1:00 Date Jan Wed Laboratory Exercise 23 Overview of the course and pre-assessment quiz Introduction/ Background-Cytokines/ Lab Safety/Solutions Lab #1: Preparation of Medium; Tissue Culture Techniques (Non-adherent Hybridoma cell line:crl1975) Assignment 1: Download MSDS forms, provide summary/ assessment; solution problems Mon 28 Lab #2: Care and handling on of mice; Harvesting of spleens, Preparation of single cell suspensions Collection of secondary anti-srbc antiserum from mice Wed 30 Lab # 2 (cont): Booster immunization of mice Lecture: Overview PCR, RT-PCR, nucleic acid purification and BLAST Assignment 1 due Problem Sets 2 & 3 Assigned: Solutions, Dilutions and Cell Counts Feb Mon 04 Lab #3: In vitro stimulation of spleen cells Problem Sets 2 & 3 due Independent Research Topic Due Journal Article #1 assigned Tue 05 Collection of culture supernatant and spleen cell lysates to be stored at -80C Wed 06 Collection of day 7 tertiary anti-srbc antiserum from mice Lab #4: Detection of TNF- expression by RT-PCR; Isolation of total RNA from spleen cells Mon 11 Lab #4 (cont): Quantification of RNA concentration & purity; preparation of cdna Discussion of Assigned Journal Article #1 Wed 13 Collection of day 14 tertiary anti-srbc antiserum from mice Lab #4 (cont): Amplification of TNF alpha by PCR & Real-Time PCR Lab #5: Set up hemagglutination of Anti-SRBC antisera Tue 14 Lab#5 (cont): Determine titer of anti-srbc on hemagglutination plates Mon 18 Lab #4 (cont): Agarose gel analysis of TNF alpha amplicons Lab #6: ELISA detection of TNF-alpha protein; Set up plates for Wed Wed 20 Lab #6 (cont): Completion of ELISA and analysis of data Grad:Literature Review for Research Proposal Due Mon 25 Lecture: Molecular Techniques-Cloning, Sequencing, PCR Primer design Problem Set 4 Assigned Lab #7: Affinity column chromatography of hybridoma monoclonal antibody; Dialysis of affinity purified mab Journal article #2 assigned Wed 27 Lab #7 (cont): Collection of affinity purified mab and determination of monoclonal antibody

2 concentration using BCA Research proposal due March Mon 04 Lab #8: Analysis of murine gamma globulin by size exclusion FPLC Discussion of Journal Article #2 Wed 06 Begin Independent Research: Bring NCBI information on cdna of interest and laptops to class Primer design & restriction enzyme analysis of cdna of choice Mon 11 Research project: Gradient PCR Wed 13 Research project: Agarose gels of PCR amplicons: set up real-time PCR Lab Journals Due: To be returned after Spring Break Spring Break March No class Mon 25 Research project Journal Article #3 assigned Wed 27 Research project Written Research Progress Report due: Discussion of progress reports April Mon 01 Research project Discussion of Journal article #3 Wed 03 Research project Mon 08 Research project Wed 10 Research project Mon 15 Research Project Written Research Progress Report due: Discussion of progress reports Wed 17 Research project Mon 22 Research project Wed 24 Research project Mon 29 Finish research projects May 01 Post Assessment Quiz Wed Exam week Research laboratory journals due Final Research Presentations Date and time to be announced Attendance Attendance is mandatory and will be taken daily starting the second week of class. Excused absences (sports related, illness, family emergencies and profession interviews) must be documented. Unexcused absences will be docked 5pts from total points for the course. 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

3 (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

4 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 800points (850 for graduate students) and will include: Midterm Lab journal 100 Lab journal for Independent Project 100 Research Proposal 100 Research Progress Report 50 Assignments and Problem Sets: 85 Journal club presentation 100 Journal Club 15pts each 75 Individual contribution/independence 40 (Graded by peers) Final Presentation of Research project 100 Post Assessment Exam 50 Total 800 Graduate Research Presentation 50 Your final letter grade will be computed according to the following scale: % of total points = A 87-89% = B % = B 77-79% = C % = C 60-69% = D <60 = F Grades will be posted on D2L 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 Midterm Lab Journal (100pts total)

5 1. Table of Contents with dates, page numbers and descriptive titles for each lab. Labs the extend over several periods subheadings with this information for each lab period. 2. Individual Lab Exercises: Note: Right and Left sides of the manual 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 Each time you complete a procedure record the following information a. Purpose (right side) b. Detailed Procedure (with buffers, dilutions, and PCR program details) (right side) c. Calculations (left side) d. Results (Including figures and graphs with figure legends) (left side) e. Discussion of results and conclusions (right side) 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. Vector maps e. 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?

6 Grading Rubric for Journal Club and Research Presentations Possible pts 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 Total (divided by 2 for Grad research presentations) 100 Score