SYLLABUS

Molecular Biological Techniques

Animal Sciences/Food Science and Technology 868

Summer (Second Term) 2006

 

July 24– August 21

MTWThF

1:00 PM to 5:00 PM

Rooms 114 (lecture) and 136 (lab) Parker Food Science & Technology Building

 

 

Macdonald Wick Animal Sciences 126 Vivian Hall 292-7516 wick.13@osu.edu Office Hrs. MWF 9:00 -10:00

Hua Helen Wang Food Science & Technology 219 Parker FST Building 292-0579 Wang.707@osu.edu Office Hrs. MWF 9:00 -10:00

 

Course Objectives

 

Upon completing the course, students will:

1.      Possess practical experience in many of the molecular biology techniques used in basic and applied research laboratories. 

2.      Be proficient in experimental design of molecular biological based questions.

3.      Be able to read and comprehend the current molecular biology literature.

 

Course Description

 

The course demonstrates the applications of molecular biology by involving the students in a real time research project.  The course employs a combined lecture/laboratory format.  Students will perform experiments using molecular biology techniques in the context of a muscle protein functionality study.  Students will identify and select a DNA sequence from the National Center for Biotechnology Information (NCBI), design oligonucleotide primers for PCR amplification of the gene, and clone the amplified gene fragment into an epitope-tagging expression vector in E. coli. Students will gain experience in protein analytical techniques by expressing the gene product encoded by the cloned DNA, purifying it by metal chelation affinity chromatography, and analyzing the expressed recombinant protein by polyacrylamide gel electrophoresis and western immunoblot techniques.  RT PCR analysis of isolated RNA will assess the expression of the muscle protein gene in various tissues.

 

Grading Criteria

 

The course will be graded on an A-E basis.  Individual student comprehension of the material will be assessed through seven homework assignments (70 %), weekly assessment of the lab notebook (10%), and an oral presentation on the last day of the course (20 %).

A         90.0 - 100

B          80.0 - 89.9

C         70.0 - 79.9

D         60.0 - 69.9

E            0.0 - 59.9

Academic Integrity (Academic Misconduct)

 

Academic integrity is essential to maintaining an environment that fosters excellence in teaching, research, and other educational and scholarly activities.  Thus, The Ohio State University and the Committee on Academic Misconduct (COAM) expect that all students have read and understand the University’s Code of Student Conduct, and that all students will complete all academic and scholarly assignments with fairness and honesty.  Students must recognize that failure to follow the rules and guidelines established in the University’s Code of Student Conduct and this syllabus may constitute “Academic Misconduct.”

 

The Ohio State University’s Code of Student Conduct (Section 3335-23-04) defines academic misconduct as: “Any activity that tends to compromise the academic integrity of the University, or subvert the educational process.”  Examples of academic misconduct include (but are not limited to) plagiarism, collusion (unauthorized collaboration), copying the work of another student, and possession of unauthorized materials during an examination.  Ignorance of the University’s Code of Student Conduct is never considered an “excuse” for academic misconduct, so I recommend that you review the Code of Student Conduct and, specifically, the sections dealing with academic misconduct.

 

If we suspect that a student has committed academic misconduct in this course, we are obligated by University Rules to report our suspicions to the Committee on Academic Misconduct.  If COAM determines that a student has violated the University’s Code of Student Conduct (i.e., committed academic misconduct), the sanctions for the misconduct could include a failing grade in this course and suspension or dismissal from the University.

 

If you have any questions about the above policy or what constitutes academic misconduct in this course, please contact either Dr. Wick or Dr. Wang.

 

Other sources of information on academic misconduct (integrity) to which you can refer include:

 

The Committee on Academic Misconduct web pages (oaa.osu.edu/coam/home.html)

 

Ten Suggestions for Preserving Academic Integrity

(oaa.osu.edu/coam/ten-suggestions.html)

 

Eight Cardinal Rules of Academic Integrity (www.northwestern.edu/uacc/8cards.html)

 

Resources for learning

Office for Disability Services

150 Pomerene Hall  

1760 Neil Ave.  

Columbus, OH 43210-1297

Phone: (614) 292-3307

24-Hour Info Line: (614) 292-0870

 

University Technology Services

http://www.osu.edu/units/uts/

The Center for The Study and Teaching of Writing

http://www.cstw.ohio-state.edu/

485 Mendenhall Labs

Columbus, Ohio 43210

(614) 688-5865

 

OSU Libraries

http://www.lib.ohio-state.edu/

 

REFERENCE LIST

 

Each student should have a copy of the course laboratory manual.  The course web site is http://class.fst.ohio-state.edu/FST868/index.html.  There is no required text.  Several books and laboratory manuals are recommended to support course material.  These will be mentioned during lecture and placed on reserve at the Agriculture Library.  Review and original scientific articles on some topics are recommended.  Most of these articles are readily available in university libraries; we will provide copies of those that are not.

 

On reserve at the Agriculture Library:

 

Textbooks:

Genes VI. 1997. B. Lewin. Oxford University Press, New York.

Molecular Biology of the Cell. (3rd ed.) 1994. B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson.  Garland Pub., New York.

Molecular Biology of the Gene. (4th ed.) 1987. J. D. Watson, N. H. Hokpins, J.W.  Roberts, J. A. Steitz, and A. M. Weiner.  Benjamin Cummings Publishers, Inc., Menlo Park, CA. 1987.

 

Laboratory Manuals:

Molecular cloning: a laboratory manual, second edition. 1989. J. Sambrook, E.F. Fritsch, and T. Maniatis.  Cold Spring Harbor Laboratory Press, Plainview, NY.

Plasmids: a practical approach. 1993. K.G. Hardy (ed.) Oxford University Press, New York.

The Polymerase chain reaction. 1994.  Kary B. Mullis, François Ferré, Richard A. Gibbs, (eds.)  Birkhäuser, Boston

PCR. 1994 C.R. Newton and A. Graham. Bios Scientific Publishers in association with the Biochemical Society, Oxford.

PCR strategies 1995 Michael A. Innis, David H. Gelfand, and John J. Sninsky (eds.).Academic Press, San Diego.

Enzymology primer for recombinant DNA technology. 1996. Hyone-Myong Eun. Academic Press, San Diego.

Gel electrophoresis of nucleic acids: a practical approach. 1990. D. Rickwood and B. D. Hames. Oxford University Press, New York.

DNA cloning: a practical approach. 1995. D. M. Glover and B. D. Hames (eds.) Oxford University Press, New York.

Recombinant DNA and biotechnology. A guide for students. 1996. Helen Kreuzer, Adrianne Massey.  ASM Press, Washington, D.C.

Recombinant DNA technology I. 1992.  Ales Prokop and Rakesh  K. Bajpai (eds.)  New York Academy of Sciences, New York, N.Y.

Recombinant DNA methodology II .  1995. Ray Wu (ed.) Academic Press, San Diego.

Methods for cloning and analysis of eukaryotic genes. 1990 Al Bothwell, George D. Yancopoulos, Frederick W. Alt.  Jones and Bartlett Publishers,  Boston, MA

Affinity chromatography: a practical approach. 1985. T.D.G. Dean, W. S. Johnson, and F.A. Middle (eds.) Oxford University Press, New York, NY

Gel electrophoresis of proteins: a practical approach. 1990. B. D. Hames and D. Rickwood (eds.) Oxford University Press, New York, NY.

DNA and protein sequence analysis: a practical approach. 1997. M.F. Bishop and C. J. Rawlings (eds.) Oxford University Press, New York, NY.

Gene transcription : RNA analysis : essential techniques.  1996. K. Docherty (ed.), Chichester

 

Internet Source Material

 

You will find many molecular biology resources on the web.  The following are a few good examples.

 

http://micro.nwfsc.noaa.gov/protocols/ (Molecular Biology Protocols)

http://www.dartmouth.edu/artsci/bio/ambros/protocols/molbio.html  (Comprehensive Protocol Collection)

http://www.public.iastate.edu/~pedro/research_tools.html

http://www.ncbi.nlm.nih.gov (Sequence database search and analysis

http://info.med.yale.edu/caim/hhmi/public/  (Laboratory safety course, REQUIRED)

 

WEEK 1

Date	Lecture	Lab
M 7/23	Introduction to course and project	Aseptic technique, microbiology and pipetting. Streak LBamp plate with E. coli
T 7/24	Basic molecular biology review Plasmid DNA isolation	Small-scale plasmid isolation (pET15b and pBlueScript) using ion exchange chromatography
W 7/25	Polymerase Chain Reaction	PCR amplification of the DNA sequence corresponding to the LMM domain of chicken skeletal fast muscle myosin heavy chain
Th 7/26	DNA quantification Agarose gel electrophoresis Basic cloning strategies	PCR product purification DNA quantitation Restriction enzyme digestion
F 7/27	Plasmid cloning vectors Restriction enzymes	Agarose gel electrophoresis of DNA samples Purification of fragments from gel
WEEK 2
Date	Lecture	Lab
M 7/30	Lac operon Blue/white selection	Run gel of purified fragments DNA ligation Inoculate E. coli for subsequent transformation
T 7/31	Advanced cloning strategy	Electrophoretic analysis of ligation reaction. Preparation of competent E. coli BL21 DE3 (pLysS) and E. coli DH5a cells. Transformation
W 8/1	Cloning vectors	PCR confirmation of ligation
Th 8/2	Degenerate primers	Plasmid isolation and purification
F 8/3	Libraries	Run agarose gel Select and prepare frozen stocks of transformed E. coli. Inoculate for protein expression.

 

WEEK 3

Date	Lecture	Lab
M 8/6	Principles of recombinant protein expression	Set up seed culture
T 8/7	Water	Bacterial growth curve Induce protein expression and take samples Freeze cells
W 8/8	Principles of SDS-PAGE	Run and stain SDS-PAGE gel
Th 8/9	Principles of stacking in electrophoresis and western blots	Protein purification
F 8/10	Principles of chromatography	Affinity chromatography Dialysis
WEEK 4
Date	Lecture	Lab
M 8/13	Principles of microarray analysis	Initiate western immunoblot analysis of expressed recombinant protein
T 8/14	DNA sequencing	Western immunoblot analysis continued
W 8/15	Working with RNA	RNA preparation from chicken tissues
T 8/16	Monoclonal antibodies	Pour and run gel for RNA analysis
F 8/17	RT-PCR	RT-PCR

 

WEEK 5

Date		Lab
M 8/20	Student presentations	Prepare and run agarose electrophoretic analysis of PCR fragments
T 8/21	Student presentations
W 8/22	Student presentations
T 8/23

 

LABORATORY POLICIES

Safety

 

·        Complete the online safety training at http://info.med.yale.edu/caim/hhmi/public/ .

·        Precautions must be taken in the laboratory portion of the course to protect us from chemicals and biohazards and to protect our experiments from microbial contaminants and enzymes on human skin. 

·        Lab coats must be worn at all times.  If you do not have your own lab coat, one will be provided for you.

·        Safety glasses will be provided for everyone and should be worn at all times in the lab.

·        When using UV light, safety glasses impermeable to UV light must be worn.  A face shield is also recommended.

·        Many procedures will require the use of gloves to protect you or to protect your experiment.  When indicated in protocol or by instructor, gloves must be worn.

·        No food or drinks are allowed in the laboratory.

·        Extinguish open flames when finished.  Never leave an open flame unattended.

·        Bio-hazardous materials must be disposed of in biohazard containers.  Solid waste goes in the biohazard box located in the lab.  Petri dishes should be taped closed before disposal.  Liquid waste goes in the waste container provided to each group to be autoclaved after class.

·        Please notify instructor if you are pregnant.

 

General

·        You will be using some expensive equipment during the course.  Please be cautious when using all equipment.  If you are in doubt about the proper operation of a machine, ASK.

·        Each group will receive 2 – 3 micropipettes.  These are expensive.  Do not drop or abuse the micropipettes.

·        Everyone will share many of the reagents you will be using.  Please take care not to contaminate reagents.  Only fresh, sterile pipettes or pipette tips should be used to remove reagents.

·        At the end of each laboratory session, clean your glassware and leave it to dry in dish drainer.  Each group will have a box in which to store their things.  Replace all items in your box when finished.

·        Sanitize your bench top before and after working.

 

Lab Notebook

An up-to-date lab notebook is an essential part of good research.  As such, each student will be required to keep an up-to-date notebook which will be collected and graded each Friday.  The notebook will be graded for the following:

 

1. Purpose and date of the experiment.
2. Experimental design.
3. ALL calculations (the notebook IS your scratch paper!!!).
4. Observations
5. Conclusions

SCHEDULE OF TOPICS

 

WEEK 1

Date	Lecture	Lab
M 7/23	Introduction	Aseptic technique, microbiology and pipetting. Streak LBamp plate with E. coli
T 7/24	Basic molecular biology Plasmid DNA isolation	Small-scale plasmid isolation (pET15b and pBlueScript) using ion exchange chromatography
W 7/25	Polymerase Chain Reaction Assignment #1: Primer design.  Due 7/31	PCR amplification of the DNA sequence corresponding to the LMM domain of chicken skeletal fast muscle myosin heavy chain
Th 7/26	Plasmid vectors	PCR product purification DNA quantitation Restriction enzyme digestion
F 7/27		Agarose gel electrophoresis of DNA samples Purification of fragments from gel
WEEK 2
Date	Lecture	Lab
M 7/30	Stokes and electrophoresis	Run gel of purified fragments DNA ligation Inoculate E. coli for subsequent transformation
T 7/31	Advanced Cloning Strategy Blue/white selection	Electrophoretic analysis of ligation reaction. Transformation
W 8/1	Lac operon Assignment #2: Cloning strategies  Due 8/8	PCR confirmation of ligation
Th 8/2	Degenerate primer design Assignment # 3: Degenerate primer design Due 8/9	Plasmid isolation and purification
F 8/3	Hybridization techniques	Run agarose gel Prepare frozen stocks of transformed E. coli.

 

WEEK 3

Date	Lecture	Lab
M 8/6	Principles of recombinant protein expression	Bacterial growth curve Induce protein expression and take samples Freeze cells
T 8/7	Principles of SDS-PAGE Assignment #4 Ferguson Plots,  Due 8/17	Prepare SDS-PAGE gels
W 8/8	Principles of western immunoblot analysis	Run and stain SDS-PAGE gel
Th 8/9	Principles of protein purification	Preliminary recombinant protein purification steps
F 8/10	Affinity chromatography Assignment #5 – Restriction Fragment Length Polymorphsim.  Due 8/17	Affinity chromatography Dialysis
WEEK 4
Date	Lecture	Lab
M 8/13	Monoclonal antibodies	Initiate western immunoblot analysis of expressed recombinant protein
T 8/14	Protein sequence analysis by MS	Image analyses demonstration Western immunoblot analysis continued
W 8/15	Working with RNA	RNA preparation from chicken tissues Quantify RNA
T 8/16	Computer resources for sequence analysis Assignment #6: Sequence analysis.  Due 8/22	Pour and run formaldehyde gel for RNA analysis
F 8/17	RT-PCR	RT-PCR

 

WEEK 5

Date		Lab
M 8/20	Student presentations	Prepare and run agarose electrophoretic analysis of PCR fragments
T 8/21	Student presentations
W 8/22	Student presentations