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BIO 422

Research Methods in Cell & Molecular Biology

The emphasis in this course is on hands-on familiarization with some of the common techniques used in molecular biology and genetics.  Each week there will be a short lecture on the physical and chemical basis for a given procedure, a description of how the technique is actually performed and then the class will go into the lab and try it out.  Each student will be expected to perform each of the steps involved; this means that some weeks you will have to come back another day to work on the technique, since there will not be time for everyone to get experience the first day.  In addition, many of the experiments will run for several days, meaning that you will have to come in each day to do the next part. Lastly, all of the solutions needed for each week will be the responsibility of the students, as will all of the clean-up.  Your ability and willingness to organize and conduct these aspects of working in a lab will be evaluated in determining the final grade.

As a part of this class you should check the library every week for journals containing papers that are using the techniques described in the course  As the class progresses you should start to become more critical in your reading of these. You may be asked to analyze such a paper as part of your exams. In addition, each student will be required to pick a protein to purify. During the course of the semester you should read about your protein, write a review paper and carry out an isolation and identification. The initial review of the protein, and a description of the proposed purification scheme are due by mid-term break. The complete write-up should follow the normal format of a scientific paper, this study will count for 25% of the grade, and is due the last day of class.

There will be written mid-term and final exams, which will deal mainly with the lecture material and together will make up 25% of the grade.

Your ability to master techniques, work effectively in the lab and to demonstrate practical ability will form the remaining 50% of the grade.  This will be determined by watching your performance on a week-to-week basis and evaluating you on the criteria listed above, along with your lab notebook.

Text: There is no required text. However, I recommend 'reading “Protein methods “ by Bollag et al., Wiley-Liss Publishers, ISBN 0-471-11837-0

Course Outline

Week

Topics

1

Measurement: spectroscopy, protein determination methods. Standard curves, pipetting, making solutions

2

Getting into cells:  osmotic shock, grinding, bead beater, French press, freeze/thaw, sonication, lysozyme, detergents

3

SDS gels, staining methods, gradient gels

4

Organelle purification:  Differential and isopycnic centrifugation, step vs. continuous gradients

5

Protein purification : ammonium sulfate precipitation, acetone, TCA, dialysis

6

Chromatography: exclusion, ion exchange and affinity.  Batch vs column purification

7

Iso-electric focussing and 2-D gels

8

Western blotting

9

Labeling and autoradiography

10-14

Independent project- protein isolation and identification


Lab Notebook:

Everything you do in the laboratory should be written in ink, in a hardbound booklet and dated.  This is your record of what was done, when, why and how.  If any questions arise about a method or the results of an experiment you should be able to find all the relevant details in the notebook.  Keeping such a notebook is a key part of this course, be prepared to hand it in at the end of the semester for examination.

Stock solutions:

These are generally solutions that fall into one or more of the following categories

           • used in large amounts

           • take a long time to prepare

           • are stable for a long time

           • can be made in concentrated form

Since some may only be made up once a year, it is a good idea to have fairly detailed instructions about the preparation.  It is easy to forget some small but critical detail.  Give each solution a short but descriptive name that is unlikely to be confused with any other stock solution.  Point out any special requirements (i.e. must be kept below 4oC, chemicals must be from Sigma), and then label the container, including pH (if applicable), date made, name of person making it and any other pertinent information.

Always used distilled water, unless explicitly told otherwise.

• Make solutions up in less than the final amount of water  Transfer to a volumetric flask for the final accurate volume.  Rinse the beaker with the remaining water and bring to the mark in the volumetric flask.

• Never use the same weighing paper, scoop or beaker for two different solutions.

• Store solution in the refrigerator, unless instructed otherwise.

For more detail on making solutions.

See: http://biology.ecsu.ctstateu.edu/courses/solutions.htm


Working solutions:

These are the solutions that you use for a specific function, i.e. pouring an 8% acrylamide gel.  Many working solutions are complex, and are often made by diluting and mixing several stock solutions and adding additional chemicals.  By and large they should be used as soon as possible, keeping overnight is usually the longest delay that should occur.  The first time that a recipe is used you should include all relevant details in your lab book, including the source of chemicals (including manufacturer's lot number).  Any changes in the composition of a working solution should be written in your daily lab book; if the changes are to made permanent then add the new recipe to the recipe book.

For a more detailed account on making solutions

Methods:

All methods should be described in detail the first time they are used.  If you are following a published protocol, then a photocopy of the method can be inserted in your lab book.  Any deviations from this method must be noted.  In later uses you can simply refer back to the relevant page.  You may wish to include a more detailed record for your own use.  Notes on the function of various ingredients may help you understand the procedure, or help you chose a substitute at some time in the future.  Remember, the purpose of this record is to allow you to come back to the book a year from now and be able to repeat the process exactly.

Laboratory Etiquette:

There are a few rules that you will have to follow if things are to run smoothly:

• Obey all safety rules; no mouth pipetting.  It is a good idea to wear a lab coat at all times and safety glasses and gloves as needed.  This also means no fooling around; what may seem a harmless joke to you may get you kicked out of the lab and a failing grade.

• If you made it dirty, clean it up.  In many cases this means more than a simple rinse under the tap.  For items like pipettes you will have to organize a system.

• If you used the last of something (or even almost the last), make some more.  Follow all directions carefully, a number of experiments may depend on the stock solution you are making.

• Be prepared to spend extra time on chores that need doing, even when it may not be your experiment that is involved.  You may need someone to do the same for you later.

ECSU Disability Statement

If you are a student with a disability and believe you will need accommodations for this class, it is your responsibility to contact the Office of Disability Services at (860) 465-5573. To avoid any delay in the receipt of accommodations, you should contact the Office of Disability Services as soon as possible. Please understand that I cannot provide accommodations based upon disability until I have received an accommodation letter from the Office of Disability Services. Your cooperation is appreciated.

Cheating and Plagiarism

A student using other than approved materials when taking a test or who gives or receives information during an examination is guilty of cheating. Plagiarism is presenting the work of others as one's own. The "work of others" includes any work bought or borrowed from another student as well as work copied from a book, magazine, newspaper, or other medium. It also includes using references that were not in fact read. Complicity in another's act of plagiarism is itself an act of plagiarism. These acts are considered academic violations and are covered by the Statement on Campus Rights and Responsibilities in the Student Handbook. Discovery of cheating or plagiarism could lead to the maximum sanction - course failure and expulsion from the University.