MOLECULAR BIOLOGY LABORATORY COURSE File: H:\BIO3151\OutlineE2001 Last Modified: June 13, 2001.

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1 MOLECULAR BIOLOGY LABORATORY COURSE File: H:\BIO3151\OutlineE2001 Last Modified: June 13, Instructors: Dr. J. Basso, GNN233: Dr. D.A. Johnson, GNN 222: This course will focus on the mechanics of DNA manipulation using recombinant DNA techniques. Because of the nature of these experiments, occasionally work will have to be completed outside of the scheduled laboratory period. GRADING SYSTEM: BIO3151 BIO8102 I. three lab reports (25,25,15) I. three lab reports (25,25,15) ii. poster (15 marks) ii. paper (15 marks) iii. lab exam (20 marks) iii. lab exam (20 marks) Several books have been placed on reserve in Morisset Library : Ausubel, F.M. ed. Current Protocols in Molecular Biology (1988) & Short Protocols, 2nd ed. (1992). QH506.C87 & QH506.S64 (REFERENCE SECTION, NOT RESERVE) Becker et al. (1996) Biotechnology: a Laboratory course. 2nd edition. TP248.2.B374 Bloom et al. (1996) Laboratory DNA Science: an Introduction to Recombinant DNA Techniques and Methods of Gene Analysis QH442.B59 Burton, Z.F. & Kaguni, J.M. (1997) Experiments in Molecular Biology: Biochemical Applications. QP519.B94 Ream, W. & Field, K.G. (1999) Molecular Biology Techniques: an Intensive Laboratory Course. QH506.R43 Sambrook et al. (1989) Molecular Cloning: a Laboratory Manual. QH442.2.M26 Winfrey et al. (1997) Unravelling DNA: Molecular Biology for the Laboratory. QH506W54 The material in this lab manual was prepared by Drs. J. Basso, L. Bonen, and D.A. Johnson unless otherwise attributed. No unauthorized reproduction of any part of this manual is allowed. SCHEDULE

2 During this course, you will be carrying out a series of experiments using restriction enzyme analysis/ gel electrophoresis to characterize an unknown DNA and to construct a map of an the unknown DNA. You will also isolate genomic DNA, use PCR to amplify a known gene and then clone it. The analysis of this clone will include the determination of its sequence and the use of the clone as a probe in Southern and Northern transfers. The sequence obtained will be later used in the section on Bioinformatics. These experiments are designed to give you hands on experience in the techniques that are routinely used in a research laboratory. WEEK 1: Introduction, course outline and objectives. Marking scheme. Forming partnerships. Use of computers and the WEB in Biotechnology and this course. Bioinformatics I. WEEK 2: Introduction, outline and objectives of the experimental system. Use of micropipettes. Restriction of a plasmid and gel electrophoresis. Bioinformatics Report I (5 Marks) and poster topic due. WEEK 3: Construction of a restriction map. Restriction analysis of unknown DNA(I) Isolation of Genomic DNA. PCR of genomic DNA with leghemoglobin primers. WEEK 4: Restriction analysis of unknown DNA(II). Gel electrophoresis of PCR product. Southern transfer. Abstract due. WEEK 5: Cloning of PCR products. Total RNA isolation. RT-PCR of RNA. WEEK 6: Poster Session (15 Marks). WEEK 7: Bioinformatics II. Finding genes in DNA. Report 1 (25 Marks) is due. WEEK 8: Transformation of ligation mixtures. RNA gel electrophoresis and Northern transfer. Purification of plasmid DNA. RT-PCR gel. Bioinformatics Report II (5 Marks) is due. WEEK 9: Screening of transformants by PCR. Isolation of insert for probe. WEEK 10: Preparation of labelled probe. Northern and Southern hybridizations. Preparation of labelled probe. Northern and Southern hybridizations. WEEK 11: Plasmid purification and quantitation. DNA sequencing. Analysis of Northern and Southern hybridizations. WEEK 12: Bioinformatics III. Using the WEB to analyse DNA. WEEK 13: Report 2 (25 Marks) is due. Bioinformatics Report III (5 Marks) is due. ATTENDANCE IMPORTANT INFORMATION

3 Please be aware of the Faculty regulations concerning absences from examinations and other tests as described on pp 4-5 in the Faculty of Science calendar. It is the policy of the Department of Biology that these regulations be enforced and that this policy be explicitly stated in the course outlines. PLAGIARISM Students should be clear as to the meaning of plagiarism. You may copy a short section of one or two lines from another work if the material copied is in quotation marks and referenced. If you copy extensively or without attribution, this is PLAGIARISM and will be heavily penalized. The penalties may include total loss of the mark. LATE PENALTIES The penalty for missing any of the deadlines for written work is 10.0% of the mark per day (weekends count as 2 days). For example, if the lab report is worth 25%, handing in the report one day late reduces your MAXIMUM MARK to 22.5%, two days late to 20.0%, etc. up to a maximum of half of your mark. The penalty for NOT preparing a lab outline and having it signed by your demonstrator is 1 mark. FLEXIBILITY Due to the nature of the experiments you may need to come to the lab outside of scheduled hours and we may need to make minor changes to the experimental protocols AT THE LAST MINUTE!!.

4 POSTER TOPICS Select one of the topics listed below. In order to reserve that topic, sign your name and your choice on the list provided outside of DRO 236. Make sure you are registered for the correct session. The poster will include both information about the technique (or molecular approach ) as well as background on the subject and its application. On poster day (your lab day) you will have 30 minutes to assemble your poster. Markers will circulate around the room and mark your poster. While waiting, read other posters and ask questions. Your poster must remain in place until 18:00 or until you are told to take them down. 5 Marks: For the poster itself, the visual impact (not too much text in small print), the flow, the information included. Can a knowledgeable person understand the topic in your absence? 10 Marks:You will be asked questions by your class for no more than 10 minutes. You will be judged on your knowledge and abilities. -DNA fingerprinting and forensic work -Analysis of DNA from ancient tissues -Antisense oligonucleotides in therapy -Antisense RNA in non-medical biotechnology -DNA diagnostics in conservation biology -DNA diagnostics in population biology -Ribozymes and gene therapy -Site-directed mutagenesis and protein engineering -Diagnostic PCR strategies-medical -Diagnostic PCR strategies-nonmedical -Genetically modified foods -Monitoring the release of GMOs -Production of human hormones -The future of DNA sequence analysis -Thermostable enzymes and biotechnology -Vaccine production by biotechnology -Biomaterials -Animal pharming -Plants as bioreactors -Recombinant protein production in yeast -Expression in mammalian or insect cells -Bioinformatics of the human genome for drug discovery -Bioinformatics and genome comparisons -Using bioinformatics to study molecular adaptations -DNA Chips for measuring gene expression -Biotechnology and Genetic Disease -DNA vaccines -Biolistic transformation systems -Biotechnology and Forestry -SNPs and designing drugs for individuals. Topic of your choice (with the permission)

5 LABORATORY SAFETY In this laboratory course, you will be using a number of different types of hazardous materials - radioisotopes, bacteria containing recombinant DNA, high voltage electrophoresis equipment, corrosive and mutagenic chemicals, etc. It is absolutely essential that proper precautions are taken at all times. In the lab manual, specific information and reminders are given at various places. 1. There will, of course, be no eating or drinking in the laboratory at any time. 2. Always wear your lab coat. When appropriate, wear plastic gloves. Be sure to remove them any time you leave the laboratory. 3. For certain procedures, it is advisable to wear safety glasses. As in other labs using volatile chemicals/biochemicals, it is not advisable to wear contact lenses. 4. Keep your work area clean and free from clutter. Place books, bags and coats in your locker, not on shelves or radiators in the lab. 5. Remember that your work habits affect not only your own safety, but that of others working around you in the lab. 6. These are intensive 6-hour labs. To be alert and careful throughout the whole period, arrange to take fresh air/snack breaks. Work with your partner as a team in order to use your time efficiently. 7. Read the lab protocols carefully before coming to the lab. Prepare flow charts. Think through how you will carry out a procedure before doing it. 8. If an incident/accident arises, inform the lab demonstrators or instructor immediately. 9. The week of the radioactive lab, there will be a presentation on the safe use of radioactivity. This information will consist of a video given in class that will supplement a Handout prepared by the Radiation Safety Officer that will be distributed prior to the class that uses radioisotopes. The proper use of radioisotopes is of utmost importance in the lab, thus, you must carefully read the information given in the lab manual prior to the lecture. To underline the importance of the information in the Manual, the video and the Handout, there will be a short test administered prior to the beginning of the class. If any student is pregnant, she should inform the instructor so that proper precautions can be taken. 10. Safety information on the chemicals used in these laboratories can be found in an MSDS ( Material Safety Data Sheet ) form.

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