Methods in Recombinant DNA Technology



Methods and Applications in Molecular Biology 11:126:427Fall 2018SyllabusCourse Director:Course Preparator:Dr. Wendie CohickRalph Dapsis108A Foran Hall194C Foran Hallcohick@sebs.rutgers.eduralph.dapsis@rutgers.edu848-932-6319848-932-6202 or 932-6193Section 1Dr. Amanda Jetzt and Dr. Cohick (Instructors)Nina Paranjpe (TA)108B Foran Hallnep38@scarletmail.rutgers.edujetzt@sebs.rutgers.edu848-932-6320Section 2Dr. Michael Lawton (Instructor)Jason Gregory (TA)222A Foran Halljason.gregory@rutgers.edumalawton@sebs.rutgers.edu848-932-6166Section 3Dr. Rong Di (Instructor)Lauren Chukrallah (TA)222B Foran Halllauren.chukrallah@rutgers.edudi@sebs.rutgers.edu848-932-6350All Office Hours: By AppointmentClass Times:Lecture: Monday 2:15-3:35 Foran Hall 138A; Labs are held in 193 Foran Hall Section 1_ Section 2 Section 3_____ Main LabTues 12:35 – 5:15Wed 12:35 – 5:15Thurs 12:35 – 5:15Follow-up LabWed 9:15 – 10:35 Thurs 9:15 – 10:35 Friday 10:55 -12:15Course Overview:This course is designed to introduce you to general techniques used in molecular biology as well as their application in research and industry. Monday lectures will be used to orient you to the upcoming laboratory, teach the theory behind the techniques you will be using, and acquaint you with additional methodologies and their applications that we don’t have time to cover in lab. You will be assigned reading to help enrich this laboratory experience.Over the course of the semester you will learn fundamentals such as cloning and DNA sequencing. With your lab partners, you will design and conduct your own experiment, including writing a research proposal, to study the regulation of heat shock protein 70 (HSP70) in a mammalian cell line. You will determine how your experimental treatments regulate HSP70 mRNA and protein levels using quantitative RT-PCR and western immunoblotting, respectively. We will also use CRISPR gene editing technology to knock out the HSP70 gene in mammalian cells. In the last laboratory session of the semester, each lab group will present their results to their classmates using a PowerPoint presentation.Required Text and Readings: There is no required text for this course. “Methods and Applications in Molecular Biology: A Laboratory Manual” by W.S. Cohick, R. Di, A. Jetzt, M. Lawton and R. Dapsis will be available electronically through Sakai. Reading to accompany lecture and laboratory material will be assigned through Sakai. Laboratory exercises are found in the manual. Please print out the entire lab manual and put it in a three-ring binder before the first lab. Be sure to read the exercises thoroughly before coming to lab.Grading ComponentPercent of Final GradeResearch Proposal10Exam #125Final PowerPoint Presentation20Exam #225Attendance and Participation10Laboratory Notebook 10Total 100100Research Proposal: Instructions and grading rubric are in the lab manual.Exams will be based on both lecture material and laboratory work.PowerPoint presentations will consist of an overall group grade (8 points) and two individual grades, one given by the instructors (6 points) and one given by your group peers (6 points).Laboratory Notebooks: All students are required to keep a detailed laboratory notebook. You need a spiral or bound “composition book” that is only used for this class – do not use a notebook that is used for other classes. You will be asked to leave your notebooks with the instructors for grading at certain times during the semester. Refer to the lab manual for instructions on how to prepare your lab notebook each week and for the grading rubric.Course ScheduleDate Topic Lecturer Sept 4-7No lecture, laboratory or follow-up lab this weekRead two reviews on HSP70 that are posted on SakaiSept 10-14Lecture 1Course OrientationCohickHSP70 and Apoptosis as it relates to research proposalPrimer Design and RT-PCR Main Lab 1Cloning Part 1: Primer Design, Generation of HSP70 cDNA by RT-PCRAssign treatments for research projectWork on research proposalFollow-up Verify RT-PCR product by agarose gel electrophoresisSept 17-21Lecture 2Cloning in Plasmid Vectors/Plasmid IsolationDi Main Lab 2Cloning Part 2: Cloning of HSP70 RT-PCR product into TOPO vector and transformation of competent cells Work on research proposalTurn in hard copy of first page of the six journal articles (PDF) you have found to support your proposal by end of main lab.Follow-upCheck plates for coloniesContinue working on research proposal. Submit first draft that covers items 1-5 and item 7 listed in the research proposal guidelines in the lab manual on page 3 to Sakai by 10 PM. Sept 24-28 Lecture 3DNA Sequencing and AnalysisLawtonMain Lab 3Work on experimental design for first 1.5 to 2 hours of labPresent overview of proposal in lab and incorporate suggested changesReceive first draft back with comments.Follow-up Pick colonies for next week’s lab.Submit a revised draft that covers items 1-7 listed in the research proposal guidelines in the lab manual to Sakai by 10 PM. Oct 1-5Lecture 4RNA analysisCohickMain Lab 4 Cloning Part 3: Plasmid Isolation and Insert Verification Revised draft will be returned with comments. Follow-up Run gels to verify cDNA inserts; set up DNA sequencing reactionsOct 8-12Lecture 5qRT-PCRDiMain Lab 5Experimental set-up and collection of plete DNA analysis exercise described in lab manual.Turn in final research proposal to Sakai by 10 PM.Follow-up No follow-up unless your group needs to collect samples.Oct 15-19Lecture 6Review Session Main Lab 6RNA analysis Part 1: Isolation, quantitation and assessment of RNA Follow-upNo Follow-upOct 22-26Lecture 7Exam #1 Main Lab 7RNA Analysis Part 2: qRT-PCR, pour gel for follow-up lab Follow-up Run PCR products on gelOct 29 – Nov 2Lecture 8Protein Gel Electrophoresis and Western Blot Analysis LawtonMain Lab 8 Repeat of experimental set-up for protein analysisFollow-up No Follow-up unless your group needs to collect samplesNov. 5-9 Lecture 9Genetic Modification using CRISPR/Cas 9 Part 1JetztMain Lab 9Protein Analysis Part 1: Quantitation of protein in cell lysatesCRISPR Part 1: Prepare guide RNAFollow-up CRISPR Nov. 12-16 Lecture 10Genetic Modification using CRISPR/Cas9 Part 2JetztMain Lab 10Protein Analysis Part 2: Protein gel electrophoresis: run gel and begin immunoblotting for Hsp70.CRISPR Part 2: Transfect cells with ribonucleoprotein complex.Work on PowerPoint presentations Follow-up Complete immunoblotting for Hsp70 Section 3 will transfect cells in follow-up instead of main lab.Nov. 19-23Thanksgiving No Lecture or LabsNov. 26 - 30Lecture 11Next Generation SequencingSnyder Main Lab 11Protein Analysis Part 3: Western Immunoblot for actin (housekeeping gene).CRISPR Part 3: Determine gene editing efficiency.Work on Power Point presentations Follow-up Lab: CRISPR; work on PowerPoint Presentations Dec. 3-7Lecture 12 No formal lecture, time for group practice for presentations Main Lab 12Power Point presentationsFollow-up No Follow-upDec. 10Lecture 13Review SessionLast Day of Classes Dec 12thSecond Exam will be given during the Final exam period as indicated on the exam schedule.Learning Goals and Measures of Assessment1. To master basic laboratory techniques and the use of standard equipment used in molecular biology studies.Assessment: Exams and evaluation of weekly performance in the laboratory (PLG 2).2. To understand the principals and applications that underlie molecular biology techniques.(PLG1).Assessment: Exams and laboratory notebook.3. To learn proper experimental design with appropriate controls (PLG 2).Assessment: Group research proposal and oral presentation.4. To learn how to formulate and test a scientific hypothesis, i.e. how to conduct hypothesis-driven research (PLG 3).Assessment: Group research proposal and oral presentation.5. To learn how to document, record, and interpret scientific data (PLG 2)Assessment: Laboratory notebook and oral presentation.6. To develop effective oral and written communication skills. (PLG 3)Assessment: Short essay component of exams, group research proposal, oral presentation. 7. To build skills required to work as a member of a team (PLG 5)Assessment: Group research proposal and oral presentation.Specific Measures of Assessment1. Two exams (short essay, multiple choice, true/false)2. Group Research Proposal: Students will work in a group to design an experimental approach that tests how a specific factor regulates expression of a given gene in mammalian cells. The written proposal will state a specific hypothesis and objectives, give the rationale behind the hypothesis based on current scientific knowledge, and describe the general methods to be used to test the hypothesis. The draft proposal will be evaluated and feedback given by the instructors on how to improve the proposal for the final version. The hypothesis will be tested by experimentation in the laboratory over the course of the semester. 3. Oral PowerPoint Presentation of Group Project 4. Laboratory Notebook (to be recorded in weekly) ................
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