CHEM 317: Biochemistry Laboratory FALL, 1996 INSTRUCTOR: Dr. D. Husic OFFICE: 303 Gessner Building OFFICE HOURS: Mondays 11:00 - 12:00 AM and 2:00 - 3:00 PM Wednesdays 12:00 - 2:00 PM Thursdays 12:00 - 1:00 PM REQUIRED Robyt, JF and White, BJ (1987) Biochemical TEXT: Techniques: Theory and Practice, Waveland Press, Inc. (Prospect Heights, IL) RECOMMENDED Efiok, BJS (1993) Basic Calculations for Chemical TEXT: and Biological Analyses, AOAC International OTHER REQUIRED A laboratory notebook is also required and is ITEMS: available in the bookstore. The notebook is to have numbered and bound pages, grids (for graphing) and duplicate pages (carbon paper). Safety goggles - Absolutely required for all experiments GOALS AND EXPECTATIONS: The main goal of this course is to introduce you to some techniques and laboratory principles used for the analysis of biomolecules and biochemical processes. The experiments chosen will demonstrate and emphasize principles discussed in detail in Biochemistry lecture (CHEM 315). I like to emphasize both the approach towards solving scientific questions and data analysis. Students in this course should continue to develop and practice laboratory safety and good technique, keep thorough, accurate and professional-like laboratory notebooks, and write concise, detailed reports using scientific journal article format. You may be asked to orally defend your data and explain the significance of your results. Thus, a good understanding of the biochemical concepts and effective oral and written communication skills are expected of all students enrolled in this course. GRADING AND EVALUATION: 1. Written Lab Reports and Other Assignments -- 50 % of the course grade 2. Preparation, Lab Technique, and Record Keeping -- 25 % of the grade. (Part of this evaluation will include observation of safe lab practices and clean-up responsibilities.) 3. Misc. Assignments and Quizzes -- 10 % of the grade. (This may include informal oral questioning/quizzing during the laboratory sessions) 4. Final Examination -- 15 % of the grade. Exam to be given on December 10, 1995. PREPARATION AND LABORATORY TECHNIQUE For each new experiment, I will distribute a handout ahead of time that introduces the experimental theory and procedures. It is expected that you read this material before the start of the laboratory period. You should outline the experimental procedure in your own words in your laboratory notebook and list the equipment and supplies that you will need for the experiment. Experiments run smoother and quicker if you are organized and prepared before coming to lab. I have designed the experiments so that they can easily be completed within a three hour lab period. At the end of the scheduled time, you will need to stop at whatever point you are at. You will not be able to come back another day to repeat or finish a portion of an experiment. Missing data due to an experiment not being completed will, of course, affect your grade. Since technique is a large portion of your grade, you must learn to be prepared and make efficient use of your time. Most experiments will be performed in small groups. Participation and cooperation by each group member is essential to complete the experiments on time. Division of labor is often a good idea. Group members must be able to work together and communicate with each other. You may meet with others in the group or with other class members to discuss the experiment and interpret the results, but your assignments and lab reports must be individually prepared. It is your responsibility to obtain all of the necessary data and enter it into your notebook before leaving the lab for the day. Of key importance in the lab: 1. Safety 2. A thorough understanding of the principles underlying the experiments 3. Attendance and participation1 4. Asking questions 1There will be no make-ups for laboratory sessions. LABORATORY RECORD KEEPING Experimental results with be collected in ink, directly into a bound laboratory notebook. All entries must be dated and I will collect the carbon pages of your notebook at the end of each lab session. Other pages are not to be torn out of the notebook. I reserve the right to inspect your notebooks at any time and you may be asked to defend the contents of your laboratory reports with data from your notebook. Standards for laboratory notebooks recommended by the American Chemical Society are included in this syllabus. It is important to leave a few pages at the beginning of the notebook for creating a Table of Contents. Alteration of experimental data will absolutely not be tolerated. The elimination of any data from a table or graph must be noted and fully justified (typically by statistical means). The discussion of the data and the explanation of data which does not correlated with the expected results is more important than having the "right answer". QUIZZES Quizzes will be given during the course of semester; you will be asked to explain concepts related to the laboratory experiments and perform calculations. These may be either oral or written and may be unannounced. WRITTEN LABORATORY REPORTS A written formal laboratory report for an experiment will be due on an assigned date, typically one week following the completion of an experiment. Late lab reports will not be accepted. All reports must be typed using proper grammar, complete sentences and paragraphs. Tables and graphs must be in ink also. Do not use pages from your laboratory notebook for your reports. Reports will be evaluated for their accuracy, completeness, format and quality (including neatness and professional looking graphs and tables). Reports that are written incorrectly or that are incomplete will be returned ungraded. In these cases, you will usually have the opportunity to redo the report after discussing the problems with me. Manuscripts in biochemical journals (not review articles) provide examples of format and content. I highly suggest that you browse through some journals in the library to see what scientific reports are like. Specific guidelines will be given for individual reports, but a general format for your reports follows on the next page. Do not wait until the last minute to write up a lab report. It is easier to start when the procedure and data are still fresh in your memory. That way, if you have questions, you will also have more time to ask me questions. For certain experiments, I will hold a help session to go through calculations, concepts, etc. prior to the due date for the report. This will most likely be on Thursdays. I will not answer last minute panic questions on the Tuesday that a report is due. The time before the lab meets is scheduled for faculty meetings and/or I will be prepping the new lab for the week. Thus, I will not be available to deal with problems of those who wait until the last minute to do their lab reports. Specific guidelines will be given for individual reports, but a general format is presented on the following page. General Laboratory Report Format Title Page - Include experimental title, date, lab section, and name(s) of experimenter(s). Introduction - The introduction should be a brief explanation of the experimental objectives and the general approach used to address the scientific question. Some theory underlying the experiment should also be included. Methods - The details of the methods should be described in complete sentences (not a list of tasks performed) only as they differ from the methods listed in the text, handout or other reference used. The source of the complete procedure should be formally referenced in the bibliography at the end of the report. - The methods section, as well as others, are written in the past tense (what was done). Also avoid "first person" usage ("I treated the samples with..."); rather, write using a passive voice ("The samples were treated with....."). Results Data: - Tables and graphs of the raw data collected are included in the Results section of a report. The textbook includes instructions on preparing graphs and tables; there are also some examples included in this syllabus. Typically raw data is used to construct preliminary tables and graphs that would not be included in a formal publication, but I would like these included in your reports so that it is easier for me to find errors should they exist. - Tables and figures should contain legends, should be neat and in ink, and should be titled, numbered, and clearly labeled (including units). Someone unfamiliar with the data and experiment should be able to remove a figure or table from a report and be able to determine the meaning and significance of the information within it without having to refer to the text of the report. Calculations: - Always show a sample calculation for all mathematical data manipulations so that I can check them for accuracy. Text: - Sometimes short explanations or descriptions of the data are included in the Results section, but analysis or interpretation of the data is not included here. Discussion - This section is a summary and explanation of the results in terms of the principles to be learned. Relate the results back to the objective(s) stated in the Introduction. You should explain how the data demonstrates what was predicted or why the data deviates from the expected or literature values. "Experimental error" is not a specific enough explanation, but one that I commonly encounter when grading. - Any questions asked in the lab handouts should be addressed in the Discussion section of the report. References - All reports should include a list of citations used for the methods, literature values, and theory or analysis included. Examples of proper citation are included below. LABORATORY RULES AND POLICIES Below is a list of rules and procedures that we all must follow for safety, efficiency, good laboratory technique and courtesy to your lab mates. General: 1. Safety glasses must be brought to each class and worn while in the lab. 2. Bench space in the laboratory is limited. Do not put coats, backpacks, or other personal belongs on the benches or pieces of equipment. They may be left on the table in Room 318. This will prevent them from being damaged by chemical spills. 3. No food or drink is permitted in the lab. 4. Be aware of the location of all safety equipment including fire extinguishers, showers, eye wash hoses, and spill control materials. Any chemical spill or personal injury, regardless of severity, must be immediately reported to the instructor. 5. Please be considerate and clean up your work areas completely at the end of the class. Return chemicals, glassware and equipment to their initial location. Do not disturb items in the laboratory that you are not using; remember that the lab space is used by others for classes and research. Remember that 25% of your grade is related to lab technique of which neatness and clean-up are a part. Each week, one student will be assigned to clean-up duty to ensure that the laboratory is completely in order at the end of the class period. 6. Do not remove any items from the laboratory. Glassware: 1. For the most part, you will be sharing glassware with students from other courses and who are doing research. Cleanliness is essential since glassware contaminants can destroy many biomolecules and interfere with experimental assays. Glassware that has contained protein solutions, organic chemicals or unknown solutions should be thoroughly washed with detergent and rinsed with excessive amounts of water. Detergents themselves will denature most enzymes, so it is essential that the glassware be well rinsed. Rinse glassware with distilled water after rinsing well with tap water. In the case of glassware that has only contained water-soluble salt or buffer solutions, just rinse well; it is not necessary to use detergent as this is often more difficult to rinse than the original solution. If using a detergent, use only the dilute Liquinox solutions which I provide. 2. Clean your own dirt glassware each lab period and return it to the appropriate place when you are finished using it. 3. Used pipettes should be placed tip up into the pipette holder immediately after use -- before materials can dry on the glass. Pipettes used for protein solutions, organic chemicals or caustic solvents should be rinsed with water first before placing them in this pipette holder. I will clean a batch of the pipettes when necessary. Please do not use any more pipettes than is necessary as the cleaning process is time consuming. Chemicals, Storage, and Solution Preparation: 1. Many of the chemicals and stock solutions that you will need will be prepared for you ahead of time. Please be sure that you know what you are doing before you begin to use these solutions, and if your are in doubt, ask me. Most biochemicals are very expensive and I only prepare enough of the reagent to complete the experiment a single time. 2. Always store a reagent as recommended in the procedure or on the bottle. Chemicals that are stored in the refrigerator or freezer must be warmed to room temperature before opening the desiccator and/or bottle to avoid condensation which may accelerate the degradation of the chemical or hydrate it so that estimation of the amount of the reagent by weight measurement becomes impossible. 3. Prepare all solutions with distilled water unless otherwise directed. 4. Label all solutions using labeling tape and markers provided. Include the contents, your initials, the class and the date when the solution was prepared. 5. If you will no longer need a solution, check with your lab partners and me before disposing of it. Some chemicals will require special disposal methods of which you will be informed. EXAMPLES OF THE TYPES OF EXPERIMENTS TO BE PERFORMED Acid/Base Chemistry Review and Buffers Solution preparation/use of pH meters Ionic Properties of Amino Acids and Proteins Titrations/pka and pI values Spectrophotometric Analyses Carbohydrates Colorimetric Determinations - Nelson's Test Glycogen Hydrolysis - Acid vs. Enzyme Catalyzed Ethanol Quantification by Enzymatic Analysis Determination of the Absorption Spectrum, Extinction Coefficient and pka for p-Nitrophenol Isolation of E. coli DNA Optimization of an Enzyme Assay for Wheat Germ Acid Phosphatase Measurement of Enzyme Activity as a Function of pH, Substrate Concentration, Time, Temperature and Protein (Enzyme) Concentration Determination of the Kinetic Constants for the Hydrolysis of an Artificial Substrate Using Computer Curve-Fitting Analyses Michaelis-Menten Kinetics Enzyme Inhibitor Effects