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LABORATORY EXPERIMENTS:

I. Introduction to the Biochemistry Laboratory

1.1 Fundamental Protein Characterization

1.2 PDB/RASMOL Computer Tutorial

II. DNA Purification

2.1 Purification of Plasmid DNA

2.2 DNA Sequence Confirmation: Restriction Enzyme Treatment

III. Protein Purification and Activity

3.1 Purification of T7 RNA Polymerase

3.2 Transcription (See Introduction to Transcription)

3.3 Characterization of RNA

IV. Genomics

4 Genetic Mutation Analysis by Temperature Gradient Gel Electrophoresis

V. Protein-Nucleic Acid Interactions

5.1 Purification of Human TATA-Box Binding Protein

5.2 Gel Mobility Shift Assay

VI. Enzyme Structure/Function

6.1 Active Site Structure of Enzymes

6.2 Determination of Enzyme Inhibition

VII. Phospholipids

7 Characterization of Phospholipid Derivatives by GC-MS

Analytical Techniques

Introduction to the Biochemistry Laboratory

Introduction to Gel Electrophoresis

Polyacrylamide Gel Electrophoresis: Proteins

Polyacrylamide Gel Electrophoresis: DNA

Agarose Gel Electrophoresis

Visualizing Proteins in Gels

Bradford Protein Concentration Assay

This laboratory course covers modern biochemical laboratory techniques and their theoretical foundations. Topics include methods for protein, nucleic acid, and lipid isolation and characterization, enzyme assays, chromatography, electrophoresis, representing and manipulating proteins and nucleic acids in 3-dimensions, and use of databases.

Instructors:

Dr. Mary E. Peek Undergraduate Biochemistry Laboratory Coordinator Office: Boggs Room 2-14 Office Phone: (404) 894-4001 E-mail: mary.peek@chemistry.gatech.edu.

Dr. Loren Williams Professor of Chemistry and Biochemistry Office: IBB Room 1309 Office Phone: (404) 894-9752 Lab Phone: (404) 894-8338 E-mail: loren.williams@chemistry.gatech.edu.

Email:

All e-mail relevant to CHEM 4581 should be sent to the Mary Peek at the address above. The subject line should be listed as student last name, student first name, CHEM 4581

Example: powers, james, CHEM 4581

Any e-mail correspondence sent to the instructors that is not in this format will not be read and will be immediately deleted.

Teaching Assistants: Office IBB Room 1203, Phone (404) 894-8338

Shelley Howerton
Melissa Kerzic
Akanksha Nagpal
Kris Woods

Textbook:

Some background reading will be assigned from Biochemistry by Voet and Voet or will be posted on this website.

Laboratory Hazards:

Some of the chemicals used in this laboratory are harmful if inhaled or ingested. Do not allow laboratory chemicals to enter your mouth or small cuts or scratches on your hands. Do not inhale powders or vapors. Do not eat, drink, or chew gum in the laboratory. Wash your hands carefully before leaving the laboratory. Always wear safety glasses. Read and follow instructions. Wear suitable clothing; sandals and shorts (unless covered by a lab coat) are not permitted in the lab.

Prerequisites:

Survey of Biochemistry (CHEM 3511) or Biochemistry I (CHEM 4511) and Organic Chemistry Laboratory (CHEM 3371) or Synthesis Laboratory II (CHEM 3380). Students must be familiar with common chemical laboratory procedures.

Grades:

Grades will be based on (1) preparation for lab, performance in lab, and laboratory notebook maintenance (25%), (2) laboratory reports (50%) and (3) cumulative exam and possibly quizzes (25%). The cumulative exam will cover any material that should have been learned from the lectures and laboratory assignments including pre- and post-lab questions.

Laboratory Notebook:

All experimental data, except instrument output, should be recorded in indelible ink in a bound laboratory notebook with pre-printed sequential page numbers. Appropriate lab notebooks are available at the bookstore. Loose-leaf or spiral-bound notebooks and loose pieces of paper are not acceptable. Students should sign each page at the end of each laboratory period. Do not leave blank pages in a laboratory notebook. A lab notebook should include protocols, identification of samples, observations, and data. Record data and observations as you obtain or make them; do not write on scraps of paper with the intention of transferring information to the lab notebook later. Do not worry if your notebook is a little messy. The recording and organization of a permanent record of laboratory observations is as important a technique to master as any of the experimental methods you learn. The research notebook is a day-by-day record of the progress of experimental work. It should reflect the integrity and honesty of the experimenter as well as the clarity of his or her thought. Hand in your notebook with your last laboratory report.

Sources of Information on Experiments:

Experiments will be performed according to instructions contained in lab handouts. Each write-up has references to Voet and Voet, in addition to a detailed procedure. You should be completely familiar with both the background material and the experimental work before beginning work in the laboratory. Before coming to lab, you should read the required material, perform the prelab, construct tables, and prepare your notebook.

Plagiarism:

Using the words of another as one's own is known as plagiarism. Plagiarism is inappropriate in this laboratory and in all other situations. Material copied from laboratory handouts, textbooks, other students, or other sources must be contained within quotes, with the source cited. Much of the experimental work in this laboratory is done in teams or groups. However all data analysis and writing should be performed independently.

Laboratory Conduct:

Equipment is the responsibility of the student using it. If you lose, destroy of abuse equipment you will be charged for replacement or repair. The balance and instrumentation areas must be kept clean and free from clutter. Report any problems with equipment to the TAs. All materials stored in the refrigerator or freezer must be capped and clearly labeled with your name, section, and contents. Keep your glassware clean.

Laboratory Reports:

Laboratory reports should be in the form of a research article. Look at articles in journals such as Biochemistry or Journal of Biological Chemistry for the general format for lab write-ups. Laboratory reports are due one week after scheduled completion of lab work. The report must be given to a TA and dated. Do not put reports in the instructors mailbox or under his/her door. Delinquent laboratory reports will be penalized according to the following schedule: 1 day late--deduct 5% of total possible points; 2 days late--deduct 10%; 3 days late--deduct 15%; 4 days late--deduct 30%; 5 days late--deduct 60%; 6 days late--deduct 80%; 7 or more days lateen credit. Saturdays and Sundays count for one day each. Laboratory reports must be printed clearly and legibly, and graphical data should be neatly presented. Follow standard usage regarding spelling, punctuation, and grammar. Sloppiness may result in point deduction. Laboratory reports must include the following:
1) A Title Page, that contains the experiment title, author, partner, date(s) experiment performed, date due, date handed in, and the author's signature.
2) Introduction, (two pages maximum) that presents concepts and hypotheses, summarizes the objectives of the experiment, and describes the general principles of any techniques employed. Cite references as appropriate.
3) Experimental Procedure, that describes the experimental details of what you did, including mistakes, accidents, etc.
4) Results, that describe the results of the experiment. Tabulate or otherwise present (neatly and appropriately) your raw data. Include examples of all calculations, and tabulate the results. If graphical presentation of data is desirable, present the graphs here. (Be sure to include both graphs and tabulated data.) State any obvious conclusions.
5) Discussion (four pages maximum), that should be an analysis of the data, explaining their significance. This section is critical to any good scientific report, and is not intended as a place to simply restate observations that the reader can get by inspection of the results. Discuss any shortcomings of the experimental procedure. State and critically evaluate any assumptions that were made. Estimate the accuracy of your results. Discuss any observations that you found unusual or unexpected, and why they may have occurred. Note and discuss inconsistencies in your data that make drawing firm conclusions difficult. Discuss improvements that could be made in the laboratory hardware and apparatus that could improve your results.
6) Questions Answer any questions asked in the laboratory instructions.
7) Criticism (Optional)--Air your gripes here! Did you like the lab? Did you learn something? Why or why not? How would you change the procedure to make the lab more interesting, less difficult, etc.?

Absences:

Lack of attendance or tardy assignments will be excused only with written documentation. No exceptions will be made to this rule.

Graduating Seniors:

Graduating seniors must identify themselves within the first two weeks of the quarter.

Honor Code:

Students are expected to follow the Georgia Tech Academic Honor Code.