CHEM 4411 / 4421 Biological Chemistry Lab I and II

Transcription

1 CHEM 4411 / 4421 Biological Chemistry Lab I and II Course Instructors Prof. Cameron Mura Prof. Linda Columbus Dr. Carol Price Intern Instructor: Ryan Lo Fall 2012 Meeting Times and Places Lecture Friday 2 2:50 PM CHEM Rm. 304 Lab Tue, Wed, or Thurs 2 6:00 PM CHEM Rm. 315 or 412 Office Hours : Monday 3:00 PM Lab Rm. 412 Friday 10:00 AM Lab Rm. 412 Professor Friday 3:00 PM PLSB 106/116 Spring 2013 Meeting Times and Places Lecture Monday 12 12:50 Monroe Hall, Rm. 130 (Tentative) Lab Tue, Wed, or Thurs 2 6:00 PM CHEM Rm. 315 or 412 Office Hours By appointment with and Instructors Teaching assistants () Catrina Campbell cac4ff@virginia.edu (Tuesday, Room 315) Jason Kuhn jpk9cn@virginia.edu (Thursday, Room 412) Kim Stanek kas4wk@virginia.edu (Thursday, Room 315) Briony Strachan bs2qe@virginia.edu (Wednesday, Room 315) Paisley Trantham pdt8ke@virginia.edu (Tuesday, Room 412) Kaitlin Bailey kw3cd@virginia.edu (Wednesday, Room 412) Required materials (both semesters): Text: Fundamental Laboratory Approaches for Biochemistry and Biotechnology by Ninfa, Ballou, and Benore. This lab manual Lab Notebook: Carbon-copy capabilities and duplicate numbered pages (you will use the same notebook for both semesters) (See your TA if you are interested in a used notebook.) Materials provided on Collab site 1

2 Syllabus for CHEM 4411 Fall 2012 Date Material Instructor Aug 31 Class time material: Lab manual pages 7-19 and 26-39; Introduction to the course video (BioLEd ; 9:50); Class time activity: Concept mapping and literature searches Ryan Lo, Beth Blanton-Kent Sep 4-6 Laboratory material: Chapters 1 & 2; Campbell.pdf (collab); How to use a pipette video (BioLEd; 12:49); general safety video (BioLEd; 2:24) Lab: 1A: Literature searches and electronic resources 1B: Pipetting fundamentals Sep 7 Class time material: Lab manual pages 40-48; CREATE method paper (Hoskins et al, Genetics Education, 2007(176): ); read and prepare Steps 1-3 of the CREATE method with Koma et al. paper (Appl Microbiol Biotechnol 2008(79):775) (Lab manual pages 40-48) Class time activity: Diagramming and analysis of data in published manuscripts Sep Laboratory material: Koma et al. paper (Appl Microbiol Biotechnol 2008(79):775) and your POI specific paper(see table pages 45-46) Lab: 2: Primary literature Sep 14 Class time material: Lab manual pages 49-55; Chapter 1 & 2; LabFax 5.pdf (collab), Preparing solutions mini-lecture (BioLEd; 12:40) Class time activity: Buffer calculations Sep Laboratory material: How to use a balance video (BioLEd; 9:44); How to prepare and ph a buffer video (BioLEd; 4:01); How to calibrate a ph meter video (BioLEd; 2:54) Lab: 3: Buffers and solutions Sep 21 Class time material: Lab manual pages 56-68; Chapters 3, 10 & 12; lactate dehydrogenase mini-lecture (BioLEd; 3:44) Class time activity: Kinetic assays; experimental design Sep Laboratory material: Chapters 3, 10 & 12; Spectrophotometer video (BioLEd; 4:52); How to use a spectrophotometer video (BioLEd; 1:32) Lab: 4: Kinetic analysis of an enzyme lactate dehydrogenase Sep 28 Class time material: Lab manual pages and 89-95; Enzyme Assays1.pdf (collab); Enzyme Assays2.pdf (collab); Michaelis Menten kinetics and inhibition mini-lecture (BioLEd; 11:13) Class time activity: Enzyme kinetics and data analysis; general cloning and transformation Oct 2-4 Laboratory material: Enzyme Assays1.pdf (collab); Enzyme Assays2.pdf (collab); How to have good sterile technique video (BioLEd; 2:18); Bacterial transformation videos 2 Ryan Lo

3 Oct 5 Oct 9-11 Oct 12 Oct Oct 19 Oct Oct 26 (BioLEd; 2:29 and 4:45); How to plate bacterial transformation video (BioLEd; 1:57) Lab: 5: Enzyme kinetics and data analysis 7A: General cloning methods and transformation NO LECTURE NO LABS - Fall Reading Days Class time material: Lab manual pages 80-88; Mazumder.pdf; Protein data bank video (BioLEd; 3:00); mini-lectures TBA Class time activity: Bioinformatics tools and methods II Laboratory material: Mazumder.pdf; BLAST video (BioLEd; 7:56) Lab: 6: Computational Biology: Bioinformatics tools and methods Class time material: Lab manual pages ; Chapters 6 & 13; Cloning and expression vectors mini-lecture (BioLEd; 10:00); Recombinant DNA and protein production video (BioLEd; 4:36); Protein SDS-PAGE video (BioLEd; 2:07) Class time activity: Recombinant protein expression and SDS-PAGE Laboratory material: How to use a centrifuge videos (BioLEd; 3:17 and 1:38); How to run an SDS-PAGE gel video (BioLEd; 4:43); How to stain an SDS-PAGE gel video (BioLEd; 5:46); How to streak a plate videos (BioLEd; 4:18 and 1:56) Lab: 7B: Recombinant protein expression and SDS-PAGE Class time material: Lab manual pages ; How to prepare a column (general) video (BioLEd; 1:20) Class time activity: Chromatography Oct 30-Nov Laboratory material: Chapters 5, 7 & 8 1 Lab: 8A: Gel Filtration Chromatography 8B: Ion Exchange Chromatography Nov 2 Nov 6-8 Class time material: Lab manual pages ; Affinity chromatography mini-lecture (BioLEd; 13:08); Affinity chromatography video (BioLEd; 0:45) Class time activity: Chromatography and dialysis Laboratory material: Chapters 5, 7 & 8; dialysis overview (collab) Lab: 9A: Affinity Chromatography 9B: Dialysis Nov 9 Class time material: Lab manual pages ; Chapters 4 & 11; BioRad assay (collab) Class time activity: Quantitative determination of protein concentration and protein-ligand binding Nov Laboratory material: Chapters 4 & 11; BioRad assay (collab) Lab: 10: Quantitative determination of protein concentration and protein-ligand binding 3

4 Nov 16 Nov Nov 23 Nov Nov 30 Dec 4-6 Class time material: Lab manual pages ; ClustalW multiple sequence alignment video (BioLEd; 5:06), minilectures TBA Class time activity: Computational biology II: Molecular visualization, modeling, & docking THANKSGIVING BREAK THANKSGIVING BREAK Laboratory material: Punta.pdf (collab); PatchDock.pdf (collab) Lab: 11: Computational biology II: Molecular visualization, modeling, & docking Class time material: Bring a print out of your figures and your computer to edit your figures Class time activity: Data and figure clinic Laboratory material: POI relevant literature; bioinformatics materials Lab: Check-out and clean lab; assembly of POI data (POI report due at 2:30 on Friday, December 7; see page 10) SATURDAY December 15, 2012, 2-5 PM: Final Exam BioLEd is the Biochemistry Lab Education Resource ( Be sure to do the pre-lab assignment for both labs; *Lecture will meet on 8/31 No lecture on 10/5 4

5 At-a-glance due dates for pre-lab assignments, assignments, and reports Pre-lab Assignments Week Assignment(s) Due this Sunday At the start Week 8:00 pm of Lab 8/26 9/2 Pre-labs 1A and 1B 1B (p 37 on 1A (p 30) 9/2 9/9 Pre-lab 2; Labs 1A and 1B assignments 9/16 Pre-lab 3; Lab 2 3 (p 53) on Assignments and Reports At the start of lab 2 (p 47) Labs 1A and 1B assignments Lab 2 assignment assignment 9/16 9/23 Pre-lab 4; Lab 3 4 (p 61) on Lab 3 assignment assignment 9/23 9/30 10/7 FALL READING DAYS 10/14 Pre-lab 6 6 (p 81) on 6 (p 81) Kinetics Report (Labs 4 and 10/14 5A) 10/21 Pre-lab 7B; Kinetics 7B (p 99) Lab 6 assignment Report; Lab 6 assignment 10/28 Pre-lab 8A and 8B 8A (p 110); 8B (p 116) on 10/28 11/4 Pre-lab 9A and 9B 9A (p 122), 9B (p 128) on 11/4 11/11 Pre-lab 10; Protein Expression/Purification Report 10 (p 141) on 11/11 11/18 THANKSGIVING 11/25 Pre-lab 11; Protein Concentration/Ligand Binding 11 (p 159) on 12/2 Protein Expression/Purification Report (Labs 5B, 7-9B) 11 (p 159) Protein Concentration/Ligand Binding Report (Lab 10) 12/2 POI Report POI Report at 2:30 pm on 12/7 12/9 Final Exam on Saturday, December, 15, 2-5:00 CHM304 5

6 Organization of Biochemistry I and II labs The first semester of this course is designed to introduce you to methods used in biochemistry. You will learn these methods while studying a protein of interest (POI) assigned to you. Unlike previous lab courses, pre-canned protocols will not be provided. This laboratory is designed for you to learn to explore, research, and investigate science independently. You are expected to read textbooks, literature, and instructions before lectures and lab sessions. The lab and lecture are designed to help you teach yourself they are not intended to passively feed you information. For the second semester (Chem4421), you will be studying the function of your POI. All of the material you study this semester is relevant to the second semester. This manual and the data you acquire are the information that you will heavily rely on in the second semester. Since each protein is different, the experiments for each will be different. In the second semester, you will design and execute your own experiments; you will also need to do troubleshooting of those experiments to make progress. Finally, you will analyze your data and, with the knowledge you have about proteins similar to yours, design a second set of experiments for that POI. Essentially, you will become an expert on your POI, a protein which has a crystal structure, but that has never been studied for function! This is true research, unlike the experiments generally performed in undergraduate teaching labs. Any characterization you are able to perform for your POI should be considered as genuine scientific progress (such as in the publications you read). Grade distribution Overall laboratory performance 100 Laboratory exercises and handouts Lab 1A and 1B assignment 25 Lab 2 assignment 50 Lab 3 assignment 10 Lab 6 assignment 50 Lab Reports POI report with Lab 11 assignment 75 Questions and figures Kinetics report 100 Protein expression and purification report 150 Protein concentration and ligand binding report 100 Pre-lab quizzes 90 (best 9) Lab notebook/pre-lab assignments 100 Final exam

7 Final Letter Grades A A A B B B C C C D < 600 F At the end of the term, the averages of each lab section will be compared to the final exam averages and normalized for differences in grading between lab sections (different ). 7