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Biotechnology Laboratory I |
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COURSE: BIOTECHNOLOGY LABORATORY I COURSE DESCRIPTION 3 CREDITS. Students become familiar with recombinant DNA techniques and gene expression. Students work with genomic and plasmid DNA, transfer, select for, identify, characterize, amplify, and purify genes. Experience with electrophoresis, polymerase chain reaction, plasmid preps and bioinformatics will be included. COURSE COMPETENCIES The student will learn recombinant DNA techniques in a context that relates to the job functions of a bioscience technician: performing tests, making products, obtaining and processing materials, controlling inventory, using and maintaining equipment and facilities, performing and documenting safe practices, complying with regulations, maintaining quality assurance, evaluating and documenting results, and communicating information. Students must be able to effectively and consistently practice aseptic technique, observe all safety rules, keep a scientific notebook, calculate and make up solutions/dilutions, practice scientific honesty, and follow procedures as they perform the recombinant DNA experiments. Students who cannot perform the critical skills of the course cannot receive a passing grade in the course.* COURSE PACING BIOT 2823 is a group-paced course. Your instructor will establish testing and assignment dates. These dates must be observed in order to maintain good standing in the class. COURSE GRADING CRITERIA A: 90-100% average SOURCE OF CRITERIA The objectives and competency levels for this course are based upon the professional judgment of Biotechnology faculty and upon comparable courses from other institutions of higher education, as well as the national skill standards for a bioscience technician. LEARNING RESOURCES Required: ATTENDANCE POLICY: You are expected to attend and actively participate in all scheduled laboratories and classes for this course. Text reading will not be a satisfactory substitute for doing the laboratory work. ACCOMMODATIONS FOR STUDENTS WITH SPECIAL NEEDS Oklahoma City Community College complies with Section 504 of the Rehabilitation Act of 1973 and the American with Disabilities Act of 1990. Students with disabilities who need special accommodations should make their request in the following way: Contact the Office of Student Support Services located on the first floor of the main building near entry 12, or call 682-7520. DETAILED COURSE OBJECTIVES 1. The student must acquire the skill of using micropipettors accurately, and know how to check the accuracy of the micropipettors. The student must know how to correctly use microfuges and other centrifuges. The student must keep a fully-documented scientific notebook (see notebook handout for details). 2. The student must be able to practice aseptic technique and know when aseptic technique is called for. The student can streak plates for isolation, do replica plating, spread plates, and can follow an SOP to prepare competent cells for transformation, using the rapid colony method and the classical method. The student knows how to track bacterial growth in a liquid culture with the spectrophotometer. The student can graph a bacterial growth curve. The student can operate a shaking water bath or dry shaker. The student knows how to responsibly handle and safely dispose of bacterial cultures and other biological materials as well as chemicals used in the laboratory. The student knows how to prepare bacterial cultures for storage at -85 degrees C. 3. The student can follow SOP and do the calculations to correctly make buffers, cultures, solutions, reagents, media, including the use of the pH meter to adjust pH as necessary as well as use of the autoclave and filters for sterilization. 4. The student can follow a procedure to extract chromosomal/genomic DNA, and can quantitate the DNA using a UV spectrophotometer and other methods. The student will use Excel and class data to generate descriptive statistics from the DNA quantification experiments. 5. The student can describe the characteristics and uses for restriction enzymes. The student can follow the procedure to perform a restriction analysis of DNA, complete with gel electrophoresis, staining, and analysis of band patterns using a computer-driven gel imaging system. The student knows how to handle restriction enzymes, DNA, and gels properly for good results. The student acquires the skill to cast, load agarose gels and run and clean the electrophoresis apparatus for good results. The student can safely and effectively use ethidium bromide for gel staining and knows how to dispose of ethidium bromide waste. The student knows the function of: restriction enzymes, compromise restriction buffers, loading dye, ethidium bromide. The student can troubleshoot electrophoresis and gels. The student can read bands in gels, including doing the measurements and calculations to estimate base-pair length of bands. The student can describe some of the limitations of gel electrophoresis for resolving fragments of similar size. The student can use a formula to estimate the range of sensitivity for detecting DNA by ethidium bromide. The student can understand the implications of linear and circular forms of lambda DNA for its electrophoresis pattern. Given the linear lambda restriction map, the student can draw restriction maps of a circular lambda genome and estimate sizes of restriction fragments in base pairs from the circular form of lambda DNA. 6. The student can explain the selection strategies for transformant colonies. The student, given the formula, can determine the transformation efficiency of an experimental procedure, and discuss what factors can influence transformation efficiency. 7. The student can explain what is meant by a "plasmid miniprep" and explain when it is used. The student can describe the purpose of the miniprep reagents in the protocol and can explain why it is important NOT to overmix and what the centrifugation requirements are. The student is aware of the availability of microcolumns for plasmid minipreps, with advantages and disadvantages. The student can examine the miniprep restriction gel and compare and interpret the lanes with cut and uncut control plasmid DNA with the miniprep DNA. 8. The student can follow a SOP to ligate two plasmids. The student can read the gel to distinguish between uncut and digested plasmids. The student can use the restriction maps of two plasmids to predict what kinds of hybrid molecules could be formed as a result of the ligation experiment. 9. The student can describe when it is appropriate to use the rapid colony transformation method versus the longer classic procedure for making competent cells. The student can explain what is meant by "seasoning" cells for transformation experiments. The student can follow the SOP to transform cells for the recombinant plasmid with dual antibiotic resistance. The student can read the plates resulting from a transformation experiment and be able to explain which are the desired transformants and why. The student can perform replica plating and calculate the % dual-resistant colonies from the partially-selective medium, and can draw restriction maps for different plasmid that could be responsible for the dual resistance phenotype. The student can describe alternative strategies for obtaining dual-resistant cells if colonies were not obtained on the LB/amp+kan plate. 10. The student can carry out the SOP for purification and restriction analysis of the dual-resistant cells, and can read the gel and describe the possible configuration of the dual-resistant cells. The student can make scale restriction maps of two of the recombinant plasmid clones. The student can discuss factors affecting the outcome of the ligation experiment. 11. The student can describe the various forms of the lambda chromosome, and the COS site. The student can follow the protocol for restriction digests of the genome for mapping. The student can interpret the gel results and make measurements to construct a graph that relates base-pair size of fragment to distance migrated for all the restriction enzyme digests used. The student constructs a restriction map of the cutting sites of the genome. The student explains why a single experiment cannot produce a definitive restriction map. 12. The student can follow the procedures to successfully create a library of the lambda genome in a plasmid vector. The student can generate maps of all potential two-fragment (vector + insert) recombinant plasmids. 13. The student can follow the procedures to transform E. coli with recombinant plasmid, and be able to explain the selection strategy for the transformants, and be able to calculate the transformation efficiency. 14. The student can describe the steps of a polymerase chain reaction (PCR) protocol and discuss the necessary attributes of the primers for PCR. The student can program and operate a thermal cycler. The student can describe the limitations of PCR. The student can describe the characteristics of the various forms of DNA polymerases used for PCR. The student will perform both a manual and automated version of PCR on a target piece of DNA within lambda. The student will perform purification of PCR product using the column chromatography method, and be able to state the advantages and disadvantages of each method. The student analyzes PCR product with electrophoresis. The student compares the advantages and disadvantages of PCR versus molecular cloning for amplifying a target piece of DNA. The student will design and carry out a PCR optimization experiment. 15. The student can follow the procedures to clone a lambda DNA PCR product into a plasmid (pBLU), doing a restriction digest and electrophoresis to confirm cutting, and then ligation. The student then uses an appropriate strain of E.coli to prepare competent cells and carries out a transformation experiment with the recombinant PCR/pBLU plasmid. The student can explain the selection strategy for this transformation experiment, and can calculate the transformation efficiency of the experiment. 16. The student will use a PCR protocol to detect an Alu insertion polymorphism in human DNA which the student has isolated from his/her own cheek cells. The student collects class data to determine the genotype distribution for the class and to calculate the allelic frequency. The student can explain polymorphism in general, and this particular human polymorphism, its significance and its location in the genome. The student works with the Hardy-Weinberg equilibrium formula and calculates a Chi-square value (using Excel) to test the hypothesis that the class as a population sample is in Hardy-Weinberg equilibrium to within a 5% level of significance. The student will carry out at least one more PCR on human DNA. 17. The student can describe methods and principles of DNA sequencing. 18. The student will design and carry out an independent investigation based on one or more of the laboratories within the course, and report on the results. The student will make oral presentations of experimental results during the mock laboratory meetings. During this course, the student will learn to perform essential job functions of a bioscience technician as defined in "Gateway to the Future: Skill Standards for the Bioscience Industry." Job Functions: Perform Tests/Assays: student will be obtaining and reading protocols and SOPs for restriction digests, gel electrophoresis, ligations, PCR, transformations. The student will prepare DNA for testing. The student will be checking equipment like electrophoresis apparatus and specrophotometers. The student will assess reagents for acceptability. Student will perform a PCR optimization test.. Manufacture Products: The student will obtain raw materials, set up equipment, do cleaning procedures and sterilizing, and make buffers and solutions. Obtain specimens: The student will obtain, label, store, assess suitability, and transport cell culture samples. Process Materials: The student will prepare buffers and reagents, organize, set up and work reactions. Control Inventory: The student will date, label, store reagents and cultures, check expiration dates and lot numbers, inform instructor if supplies are low. Maintain Equipment/Facility: The student will check calibration of micropipettors and some instruments, validate experimental procedures, perform equipment maintenance, clean the work area. The student will maintain centrifuge logs and observe procedures for the safe use of instruments. Observe and Document Safe Practices: The student will maintain and follow the chemical hygiene plan, follow universal precautions for biological pathogens, practice aseptic technique with bacterial cultures, use protective equipment, observe rles of electrical safety, know the location of and use of MSDS, fire extinguishers, chemical shower, eyebath. Comply with Current Accreditation and Government Regulations: The student will follow the safety rules of the laboratory. Maintain Quality Assurance: The student will check, verify integrity of product, procedure, specimens, use controls, and follow policies and procedures.. Evaluate, Document, and Report Results: The student will collect data, perform calculations, use Excel to do statistical analysis, evaluate validity of results, and identify abnormal results. Communicate and Document Information: The student with interact with instructors and peers in the laboratory orally, coordinate tasks with peers, keep a scientific notebook, make oral presentations of results at "lab meetings," and report results of an independent investigation. The student will use a DNA database and use computers for statistical analysis and gel imaging. The student will notify instructor about problems and observations. Perform Initial Research: The student will design and then perform a research protocol and maintain a laboratory notebook. Care for Research Organisms: The student will monitor the growth and viability of E. coli cells and will prepare cultures for longterm storage. Maintain Professional Competency: The student will read and report in writing about the background history of some of the science on which the course experiments are based. The student will be able to obtain necessary information independently using the library or the internet. The student can function independently and as a member of a team. The student will be encouraged to participate in training others, as time allows. The student will conduct herself/himself as a professional while working in the laboratory. *CRITICAL SKILLS: The student cannot earn a passing grade in the course unless he/she can effectively
and consistently: The scientific equipment that students will use and become familiar with during
this course includes: |
Oklahoma City Community College Biotechnology Program
Dr. Fabiola Janiak-Spens, Program Director
7777 S. May Avenue
Oklahoma City, OK 73159-4444
(405) 682-1611, ext 7414
Comments: Fspens@occc.edu
Copyright 1998 Oklahoma City Community College
