Professor

Neal Woodbury

PSD-105

965-3294

NWoodbury@ASU.EDU

Teaching Assistants

Arlene Haffa	almhaffa@imap3.asu.edu
Matthew Rosenow	Rosenow@asu.edu
Diane Hu	diane.hu@asu.edu

Welcome to Biochemistry 467. This laboratory manual describes the experiments you will perform during this course. You will notice that, unlike previous labs you may have taken, you are not given step by step procedures. Here, a great deal of what you do you will have to think through on your own. The procedures you are given are general procedures, such as you might find in a technical manual or in a library reference. In some cases, you will have to adapt these procedures to the particular experiment being performed or the equipment which is available for your use. You will also sometimes be given updates to parts of the manual as the semester goes on. Depending on how one experiment works out, we may need to modify the next somewhat. This less directed approach comes as something of a surprise to some students, but it is much more like a real-life laboratory situation.

Web Site and email: A web site has been constructed that contains an annotated copy of the manual and will be used for urgent, timely announcements as well as for data sharing and other instructional activities. It can be accessed through http://www.public.asu.edu/~laserweb/Woodbury/classes.htm . Students are encouraged to communicate with the instructors of the course via email. We ask all students to send an email message ASAP to NWoodbury@asu.edu with CHM467 in the subject field. In this message, just say who your TA is and we will make up sets of email files that can be used to send up to date information directly to you. This way if the results from one lab period force us to change the procedure for the next period, we can contact you quickly. If you do not have an email account, please get one at the computing commons.

Class Procedures. This is the biochemistry laboratory for biochemistry majors and chemistry majors with a biochemistry emphasis. In this class, you will be introduced to common techniques, experimental theory and experimental problem solving in the framework of a biochemical laboratory. The class meets twice a week. On Monday there is a lecture coving the week's experiment from 12:40 to 1:30 in PSA113. On Wednesday from 12:40 - 1:30 we will also meet in PSA113 to discuss bioanalytical methods in a more general sense (see below). The laboratory practice itself will occur on Weds. from 1:40 to 6:30 pm in PSH431. Attendance is mandatory at both the Monday lecture and Wednesday laboratory sessions each and every week. If you are ill, please notify one of us or a TA as quickly as possible, and we will try to work out a substitute assignment (there is normally no way to make up a lab). An unexcused absence for either the Monday lecture or the laboratory meetings results in a zero for that week’s laboratory. Three excused absences will result in an incomplete for the course and you will have to retake it. The Monday meetings are very important, as will be described below. If a student makes a habit of being tardy, they will not be allowed to enter the laboratory when late and will receive a zero for that lab. Be here, be here on time, and be prepared.

On Mondays, the principles, techniques and safety aspects of the upcoming laboratory will be explained. This is extremely important. You will get little out of the lab if you do not understand what you are doing and if you are not prepared to do it. You will be given handouts with laboratory procedure updates on Monday as well. You are expected to be ready to perform those procedures without delay in your lab section. This means you should already know how much of the different reagents you will need to use (whenever possible) and have considered how you will measure them and what you will put them in once you have measured them. In order to facilitate the preparation process, you will be given a series of questions to answer for each laboratory that will be handed in at the beginning of the lab period. These preliminary questions will be a significant part of your grade (see below). If everyone comes prepared, the labs can be finished in the scheduled period.

Laboratory notebooks. All students must keep a laboratory notebook. We recommend the large (8.5 by 11 inch) notebooks that are ruled in both dimensions to make graphing easy. You will not actually turn the notebook in, but we will look at it frequently during class to make sure you are prepared and are recording the appropriate information.

Laboratory reports. Each week you will turn in a laboratory report. Reports are always due in Lab (Wednesday) at the BEGINNING of class. Late reports will be only worth 50%. NOTE: A report is considered to be LATE if it is not handed in at the BEGINNING of the lab period! Your reports will be prepared on a word processor. Graphs can be prepared by hand, though we strongly suggest that you learn to use a graphing program if you have not done so already.

For each solution or material that you prepare (e.g. a buffer, a polyacrylamide gel, a chromatography column) report:

1. How much of each substance did you measure out?
2. What form were the substances in (solid, liquid, slurry, etc.)?
3. What order were things added in?
4. Did you have to dissolve something?
5. Did you have to heat or autoclave something, and if so what conditions were used?

For each reaction that you run, bacterial growth you initiate, measurement you take or separation procedure you perform please report:

1. What components were added, in what order and in what amounts?
2. What temperatures were reactions run at, organisms grown at or measurements/procedures performed at?
3. What were the major instruments you used (e.g., water bath, 37 C growth chamber, ultracentrifuge, spectrophotometer, etc.) and what conditions were they used under (e.g., water bath temperature, centrifuge speed, spectrophotometer wavelength range, etc.)?
4. In the case of measurements, what were the raw readings that you took (these can be put into tabular or graphical form, as appropriate)?
5. In the case of reactions, growth experiments, or isolations, describe the appearance of the sample at each step (solid, liquid, clear, opaque, red, green, viscous, etc.).

In this section of the report you should be thorough, objective and well organized (data should be in tables or presented as graphs as appropriate). Do not attempt to interpret your data or observations at this stage, simply record them. You might think of this part as a description of what happened as recorded by a video camera. Video cameras only record what occurred, not what they think occurred. You grade for this part will be determined largely by how well you follow the instructions above. Your TA's will use these instructions as the criteria in the grading.

In the second section of the report (the Interpretations section), you will start at the beginning of the experiment again, but this time describe in a step by step manner what you thought was happening at each point in the experiment and explain your reasoning which led to this interpretation. You need to write down what happened throughout the laboratory from beginning to end, just as above, but this time do it from the perspective of a chemist (instead of a video camera) saying what you think is going on in the test tube at each point. This is narrative-style report. It should be written in the third person. You do not need to repeat a detail procedure, just refer to the place in the procedure you described above and describe what you think was occurring. For example, at a centrifugation step you might write "When the lysed cells were centrifuged, the larger, more dense debris pelleted. This probably included cells that did not lyse and large fragments of cell walls. The less dense, soluble components such as the nucleic acids, sugars, small membrane vesicles and soluble proteins remained in solution. The purple-brown color of the supernatant was presumably due to the bacteriochlorophyll and carotenoid containing protein complexes observed previously in the absorbance spectrum…" Note the difference between this section and the last section. We cannot see lysed cells. We cannot see that the soluble proteins are less dense than cell walls. We cannot see that it is the carotenoid and bacteriochlorophyll molecules that make the solution purple/brown. These are suppositions based on our knowledge about the system and about chemistry in general. You should describe any chemical reactions occurring. Here you should also include any calculations or analysis based on models (e.g., log plots of molecular weight vs. mobility in a gel, or calculations leading to the determination of the concentration of a protein). Such information should be presented in tables or graphs, as appropriate.

In this section you will again be graded on how well you follow these instructions, and on how clear your logic and explanations are.

In the third and final section of the report (the Questions section), you will answer a series of questions that will be provided to you.

Note that near the beginning of this manual is a sample laboratory along with a sample prelab and lab report. Follow these examples carefully.

Safety. Specific safety procedures will be outlined on Mondays as appropriate for the upcoming experiment. Certain practices should be observed at all times. Safety goggles must be worn at all times by all persons in the laboratory. This is a state law. There will be no exceptions. If you do not have goggles, they can be purchased on the first floor of H-wing (if yours are scratched up, get a clean pair -- it’s well worth the few bucks). You must have purchased these goggles before the first lab or you will not be allowed to enter the laboratory. No goggles = No lab = Score of zero. Bring your goggles every time! There is no eating or drinking allowed in the laboratory (another state law). If you are concerned at all about your clothes, I suggest you purchase a lab coat from the bookstore. We will use many reagents in the laboratory designed to stain proteins. Most of your clothes are made of proteins. These stains will never come out. Disposable gloves are provided in the laboratory and should be worn when using water soluble toxins (note that many organic solvents go through these gloves). Please avoid wearing open-toed sandals in the lab.

Bioanlaytical training. For the first time this year, this course will be worth 3 credits instead of 2. The extra credit will be for an additional hour of bioanalytical training that is added at the beginning of the lab period (12:40 to 1:30). A room for this is being arranged. During this hour, we will discuss the general principles of common laboratory techniques in some detail such as chromatography, centrifugation, electrophoresis, etc. This information will focus mostly on techniques we actually use in the course but will also be used to introduce techniques that we do not have the time to perform in the laboratory. There will be a final exam based on this aspect of the course.

Grading. The grade for this course will be based on four things. Your laboratory write-ups will be worth 60% of the grade. Another 10% of the grade will be based on your answers to the prelab questions that you hand in at the beginning of each lab. Additionally 5% is based on your performance in the laboratory including organization, promptness, preparedness, concern for safety and general attitude (assigned by the TAs). 25% of the grade will be based on the final exam covering the bioanalytical part of the course (see above).