LABORATORY COURSE IN BIOCHEMISTRY AND MOLECULAR BIOLOGY
BIOCHEMISTRY 421: Fall 2010
OBJECTIVES
This course is designed to provide you with an introductory experience to conducting experiments in a biochemistry laboratory. You will be learning a broad spectrum of modern techniques and their underlying physical, chemical and biological principles. Biochemical tools and concepts are at the core of recent advances in medical, forensic and agricultural sciences that have improved our lives over the past 30 years. The pace of biochemical research is brisk and very competitive, and innovations and new techniques appear almost daily. Interestingly, the basic ideas and strategies behind new technologies are often preserved from established methods. What you learn in this course will be immediately useful to you whether or not you choose to continue in science. You will gain problem solving skills as well as new insights to biological processes that will help you make your way in “the real world”!
The main objectives of this course are to:
1. become familiar with methodologies and instruments usedin biochemistry laboratories.
2. relate and apply these basic biochemistry skills to your chosen field of interest.
3. learn how to collect, record and analyze experimental data.
4. present results clearly in graphic, tabular, and written formats.
5. perform experiments in an environment requiring teamwork.
COURSE FORMAT
You will work with lab partners for the duration of the course. All of the experiments are designed with this in mind. You should choose partners during the first lab session.
Each lab will start with a prelab lecture in ISB 329. Lectures will start promptly at 8:30 AM. The lab section (ISB 268) will immediately follow the lecture.
Bring a calculator to lab each week.
Experiments to be performed in Biochemistry 421 are contained in this manual. The appropriate background information contained along with the experimental procedures should be read prior to the laboratory period in which the experiment will be conducted.
RECORDING DATA
All essential data are to be recorded in your laboratory manual- you are not required to obtain a separate laboratory notebook. These include but are not limited to protocols, identification of samples, and all primary observations. Do not write on scraps of paper. Write everything down clearly. Remember, sometime later you (or someone else) might have to go back and rely on your notes to figure out what you did!
ATTENDANCE
The class meets from 8:30 AM to 12:30PM on either Tuesdayor Thursday (depending on your section) as indicated in the syllabus. Because the lab schedule is tight, class begins promptly! Please be on time; latecomers disrupt the lecture.
Attendance is mandatory. There will be no opportunity to make up missed labs. One unexcused absence is grounds for being dropped from the course. Serious, unavoidable conflicts (including religious observances) should be brought to the attention of the instructor one weekprior to missing the laboratory period. Extended absences due to illness, sports events, ROTC exercises may result in withdrawal. Any student requiring special accommodations MUST contact the instructor in the first two lab periods.
POLICY ON PLAGIARISM
Plagiarism covers a range of misrepresentations, many of which are easily avoidable by proper use of citations and good writing skills. The academic honesty policy at the University of Massachusetts Amherst defines plagiarism as: "...knowingly representing the words or ideas of another as one's own work in any academic exercise." Please refer to the following website for these policies:
For this class, specific examples of plagiarism include:
1)failure to provide a citation (reference) for data/information obtained from another source.
2)directly quoting from a cited source without using quotation marks.
3)closely paraphrasing a cited source.
4)using the same or very similar wording as another student in the class, past or present.
Plagiarism is not acceptable and will result in NO credit for the assignment and notification of the Academic Honesty Board.
LABORATORY CONDUCT
1. Much of the equipment used in this course is expensive and none of it is insured. If you have trouble with the operation of an instrument, do not hesitate to ask the instructor for help.
2. Never bring food or beverage into the laboratory. Do not dispose of food wrappers or beverage containers in the lab wastebaskets, even if the contents were not consumed in the lab.
3. Work safely in the lab. You are required to wear eye protection in the laboratory at all times. Wear proper eye protection before turning on any UV light. Place backpacks, coats, jackets and sweaters in the available shelf space to avoid fire and chemical spill hazards. Note the locations of First Aid kits, fire extinguishers, and drenching showers.
4. Always think ahead. Prepare for the next step in the procedure while waiting.
5. Make sure you understand what you are doing. If you do not, feel free to ask.
6. Keep your bench area, balances and instruments clean and clutter-free. Wash your hands before leaving the lab.
7. Cell phones and music headsets cannot be used in the laboratory.
Additional note: when an experimental procedure calls for addition of water, you should only be using sterile distilled water, provided in bottles.
Assignments and Grading
Attendance is required. As such you will receive credit for attending lecture/lab on time; you will not receive credit if you are late to lecture or leave early from lab.Data assignments are due at the end of labon all twelve lab sessions. These are mostly data entries into your lab manual, calculations performed upon data or questions to be answered in your lab manual. Show your work to the Instructor before leaving class for credit.
Lab Reports (LR) are due at the start of the lecture period (8:30 am SHARP) on the lab dates indicated. Lab reports should be handed directly to the instructor at the start of the lecture. Lab reports will not be accepted at any time during the laboratory. There are three lab reports and one data report to be written during the semester. You may submit only three lab reports and the kinetics data report for credit.
You and your lab partners may turn in joint lab reports for which you will receive the same grade. On the cover sheet of the report you must each sign a statement to the effect that all partners contributed equally to the preparation of the report. You may not turn in a joint lab report with anyone else in the class.
Grading Plan
12 Attendance/Data 10 pts each 120 pts
3 LRs100 pts each 300 pts
1 Data Report 80 pts 80 pts
There are 500 points in total for Biochem 421.
Assignment due dates/times and point values subject to change by the Instructor with advance notice.
Biochemistry 421 Fall 2010 Tentative!
Tuesdays/Thursdays
September 7/9Introduction to the course, lab tour, and safety training
Micropipetting and calculations!
September 14/16
Serial dilutions, principles and methodology of ELISAs
Perform an ELISA and analyze results to model disease spreading in a population
September 21/23
Methods to determine concentration/purity of protein and DNA
Create standard curve and determine protein concentration by BCA assay; quantify DNA
September 28/September 30 (LAB REPORT ONE DUE)
Affinity Chromatography
Purify protein and determine enzyme activity by assay
October 5/7
Principles of SDS-PAGE
Run SDS-PAGE gel of affinity chromatography samples, quantify protein by BCA
October 12/14
NO LABORATORY THIS WEEK: TUESDAY IS A MONDAY!
October 19/21
Gel filtration to separate biological molecules
Perform gel filtration to separateanimal hemoglobin, myoglobin, and cytochrome c
October 26/28 (LAB REPORT TWO DUE)
DNA methods in molecular cloning: transformation, selection, plasmid vectors
Transform E. coli with DNA and plate on selective medium
November 2/4
Introduction to PCR and uses in genetic characterization and gene expression
Prepare DNA from foods and set up PCR reactions; count bacterial colonies
November 10/11 (NOTE: THURS. SECTION MEETS ON WEDNESDAY!)
Plant stress, RT-PCR, and DNA gel electrophoresis
Perform plant stress experiment
Run, photograph, and analyze gel to determine if food is GMO
November 16/18
Open class discussion
Run RT-PCR reactions on gel and determine stress response
November 23/25
NO CLASS- THANKSGIVING
November 30/December 2 (LAB REPORT THREE DUE)
Enzyme Kinetics
Determine reaction velocities; calculations/graphing to determine Vmax and Km
December 7/9 (KINETICS DATA REPORT DUE)
Enzyme Inhibitors
Characterize inhibitors and in-class discussion of data