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Molecular Biology Laboratory Course - 188

Molecular biology majors will normally enroll in MOBI188 (Molecular Biology Laboratory) in the second semester of their junior year. MOBI188 is taught in the spring by Dr. Tina Negritto and it provides students with hands-on experience on many molecular biological tools used in the context of a real research project.

Molecular Biology 188 Course Description

Objective

The objective of this course is for you to experience the process of scientific research, from defining a problem, to designing and conducting experiments, to drawing conclusions from your own data and proposing future experiments. You will be involved in investigating an important cellular process by using fundamental molecular biological techniques and approaches.

Course Organization

MOBI188 is a laboratory course that will provide you with hands-on experience, for an entire semester, on many molecular biological tools used in the context of a real research project. This means that the project you will be working on is part of an ongoing research project that nobody has done before and your results will hopefully contribute to a better understanding on the topic and lead to new discoveries.

The cellular process investigated in this course is homologous recombination, particularly identifying genes involved in the control of short sequence recombination (SSR) and how they function at the molecular level. To determine if a factor has a role in the control of SSR, my approach is to first build a strain with a null allele of the gene of interest and test its recombination phenotype. Additionally, in order to look further into the molecular mechanism of homologous recombination, the proteins involved in this process can be localized to recombination intermediates by using chromatin immunoprecipitation studies.

In the first part of the semester you will become familiar with the research project and system used by reading and discussing in class assigned scientific papers as well as papers suggested by you. We will start right away learning methods in molecular biology and undertaking experiments proposed by the instructor. As you gain more insight and confidence working in a molecular biology laboratory you will be able to work more independently and undertake a couple of different projects.

By the end of the semester you will have experienced what it is like to work in a research lab, dealing with a real research project, learning to troubleshoot, to organize yourself, to be efficient, multitask and to analyze and interpret the results obtained. In addition, you will have learned important molecular biology techniques by actually carrying them out in the lab. Other methods in molecular biology not undertaken in lab may be introduced in classroom discussions. In addition, molecular biology faculty will visit us and present a description of their research projects and systems used, as examples of the broad use of molecular biology for the study of a wide range of biological processes.  This will help you identify topics of interest that you can pursue later as part of a summer research program (highly recommended) or as your experimental thesis in your senior year.

Molecular Biology 188 is a full credit course. We will meet two full afternoons per week (Thursday and Friday). Attendance at every class is mandatory. Classes will be a combination of short lectures and bench work. You are expected to put in extra time each week in the lab, in addition to Thursday and Friday afternoons. You are responsible for completing the necessary work in between classes, which is essential in order to be able to execute the experiments scheduled for each class. This time is in addition to that spent reading, thinking, and planning experiments. For example, some weeks the extra lab time may involve multiple relatively short sessions (starting cultures, making streaks, setting up PCR runs, etc.); some others may require a more significant amount of time, especially if an important experiment needs to be repeated, and this is highly probable when doing research. Please do not leave class until you understand exactly what it is that you need to do in between classes. In this class it is really hard to keep a strict calendar of what we are going to be doing each week. You have to learn to be flexible and adapt to how things are working out in lab, since it is real research. Sometimes things go wrong and need to be worked out before we can continue. It is essential that you take ownership of the projects you are working on and work hard at pushing them forward. Ten percent of your grade is determined by Conduct and Participation, which includes how well you are prepared for class, how your projects advance and your ability to work effectively outside of class.

Lab Notebooks

You need to keep a bound notebook, which will be collected and graded twice during the semester. You have to record everything you do and in such a way that somebody else could repeat the same experiments and most important, get the same results.

In your notebook each experiment should include the following:

  • Objective
    A short explanation of what you are trying to accomplish and how (10 points). What is your hypothesis, what are the expected results or possible outcomes. (10 points)
  • Protocol
    A detailed explanation of what you did and how (10 points). Remember, if you make any changes to your protocol make sure you annotate them. For example, volumes used, type of materials used, reagents used, incubation times and temperatures. Include all details that are relevant. Make sure to include units on all values reported in your notebook (10 points).

    Each section of your notebook should have the date you did that particular experiment, and on what page that experiment continues. Protocols should be included in your notebook and when referenced later on. Make sure to include the page number where it can be found. (10 points)
  • Results
    Make sure your results are presented in a clear, neat and organized manner (10 points) . Record the actual data you obtained; for example, pictures of gels, plates, or Southern blots: cut, paste into your notebook, and label. Include important information such as conditions used or any information on variables that can affect the outcome of the experiment. (20 points)

  • Conclusions
    Make sure you always write a conclusion after you have done an experiment. Did you get what you were expecting? What do you conclude from the data obtained? Add comments on how you believe you can improve this particular experiment or what is your next step. If it did not work, discuss what you think went wrong, what can you change, did you realize what you did wrong? (20 points) Do not feel embarrassed or bad about mistakes, we all make them. The important thing is to try not to repeat the same mistakes or conditions that you know will not work.

Final Lab Report

At the end of the semester you will be turning in a report that includes all the work you have done in the semester, written in scientific format. Guidelines given in Bio40 or 41C lab manuals are relevant reading. More specific guidelines will be discussed in class.

Lab Conduct

LABEL EVERYTHING. Eppendorf tubes should be labeled on the cap and with a labeling tape on the side of the tube. Always use permanent markers. Plates are labeled on the bottom (not on the lid). Culture tubes are labeled on the tube, not on the cap.

DO NOT THROW AWAY clones or DNA or strains, streaks, etc. until we are absolutely sure we will not need it any more or that it is stored in our freezer stock.

CLEAN UP after yourself. Make sure you do not leave a mess at the end of the day. Put pipettors away in your assigned drawer.

Take aliquots of stock solutions. If you see that a supply is getting low, let me know so that I can order more before we run out completely

Enzymes are delicate and expensive. Always keep them in the enzyme box, replace the lid on the box as soon as you are done and put the box back in the freezer. Do not hold the enzyme in your hand for long and just grab the tube from the top minimizing the contact of your fingers with the bottom of the tube where there is enzyme. Always use a new tip when trying to get an aliquot out.

The water to be used in this course is in a big carboy that says dH20 MolBio. Use only this water or the small 100 ml bottles with sterile water in each bench.

Remember to be a good team player, cordial, respectful and helpful to others. We are all here to learn and get the most out of this lab experience. There is no such thing as a stupid question.

Safety

You are to wear a labcoat and safety glasses at all times while in the lab. No food or drink allowed.

The best way to avoid problems is to work carefully and think about what you are doing. Before starting a new protocol, check what chemicals you will be using. Are you wearing the appropriate protection (goggles, mask, labcoat, gloves, glasses, etc.)? Is there a chemical you are not familiar with? There are Material Safety Data Sheets (MSDS) for all chemicals in big binders in the lab. Please make sure to read the appropriate MSDS sheets before you start working. Make space in your bench. Avoid clutter in order to minimize the chances of spilling something. Working on a crowded bench can lead to bumping into things and tipping over things; make sure you work comfortably. Do not work on top of your lab notebook!!

  1. Waste must be disposed of properly. Petri dishes with nasty growing things (or beautiful growing things, depending on your taste) should go into the biological waste bin (red bag). Phenol:chloroform should be disposed of in the container that says "Phenol:chloroform Waste" in the hood (empty your eppi tubes into this container and throw empty tubes into bin for used tips disposal in hood). If you are not sure how to dispose of something please ASK!
  2. No open toe shoes and no shorts in lab.
  3. Always wear gloves when handling ethidium bromide, acrylamide, phenol:chloroform or other hazardous chemicals.
  4. Never, never, never, mouth pipette.
  5. You will be getting the access code to the lab. You may come to lab at your own convenience, but as a matter of good safety practice, do not come alone at night or on weekends. Bring a lab partner or a friend. DO NOT leave the front door of the building open (all molecular biology students will have card access to the building) and lock the lab door behind you (regardless of which side of it you are on).
  6. Oh! By the way Never, Never, Never, mouth pipette.

Grading and Due Dates (Sample)

(A more specific syllabus with deadlines will be handed out in class.)

Assignment Points Due Dates
Genomic tagging homework(Southern and diag PCR) 75 TBA
homework to be determined 75 TBA
Design digest of genomic DNA for Southern blot 75 TBA
Presentation 1 0 TBA
Quiz 1 75 TBA
Quiz 2
100 TBA
Notebook first half 100 TBA
Quiz 3
100 TBA
Final Report 200 TBA
Notebook second half 100 TBA
Lab conduct and participation 100 TBA

Participation refers to your involvement in class discussions, your overall effort in the lab, how well you prepared for each lab and your ability to work effectively outside of class hours.