CREIGHTON UNIVERSITY
CHM 382: Fundamentals of Biochemistry Laboratory
2:30 PM - 3:20 PM, Prelab Lecture Monday, HL 246
1:30 PM - 4:20 PM, Lab Wednesday, RS107
Fall 2009
INSTRUCTOR
Dr. Juliane Soukup
Rigge 233, Phone 280-3265
Email: jksoukup@creighton.edu
Website: http://chemistry.creighton.edu/~jksoukup (you will find class notes & other handouts on my website)
INTRODUCTION
Welcome to Biochemistry Lab! In this semester you will be introduced to the standard and current
methods used in biochemistry laboratories. You will gain an appreciation for the techniques that (1) are
utilized to purify and characterize biomolecules, (2) that investigate chemical reaction kinetics, and (3) that
are performed in genetic engineering.
This syllabus contains course information you will use throughout the semester. Please take a few minutes
to familiarize yourself with its contents before our first laboratory session. If you have questions regarding
course organization or policies, please ask.
As your professor I wish you success in this course, and I am here to help you whenever you need it. My
best advice is to stay on schedule and come prepared to each laboratory!
OBJECTIVES
You will be introduced to some of the basic techniques used in biochemistry laboratories. You will be able to
define and describe these biochemical techniques and understand the theory behind each one. You will
apply this knowledge to write coherent laboratory reports in which you analyze your experimental results.
You will also be introduced to scientific literature. You will read articles and critically review the findings.
ATTENDANCE IS MANDATORY!! There will be NO opportunities to make up laboratories therefore you
must be present for every lab and pre-lab meeting!
OFFICE HOURS
The times I will be in my office are listed below. Other times are available by appointment.
Tuesday: 12:00-1:00
Wednesday: 9:30-10:30
Thursday: 9:30-10:30
REQUIRED TEXT AND ACCESSORIES
TEXT: D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, 5th edition. Chapters to be
discussed are listed on the attached tentative schedule.
CALCULATOR: NON-PROGRAMMABLE scientific calculator. No programmable calculators will be
permitted to be used for any exam or quiz.
GOGGLES
LABORATORY NOTEBOOK: with duplicate pages (purchase in chemistry stockroom)
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QUIZZES
There will be a midterm and a final quiz during the semester. Quizzes will be closed book, closed notes. The
format of the quizzes may include multiple choice, problem solving, matching, true/false and short essay.
The dates for the quizzes are tentatively scheduled on the calendar. The quizzes are worth 50 points each.
An official document (physician’s certificate, etc.) is necessary in cases where it is physically impossible for
you to attend a quiz. You must notify me beforehand to take a quiz at any other time than when scheduled.
The alternate quiz will be different, more difficult, and may be given in a written or oral format. Regardless of
circumstances, only one quiz may be made up. You must take a makeup quiz within three days of the time
originally scheduled.
There will be a one week limit, from the day the exams are returned, for regrading. Your concerns must be
submitted in writing and in addition, the entire exam will be regraded, not just the answer in question.
LABORATORY NOTEBOOKS
Laboratory notebooks will be completed individually. The due dates for these items are indicated on the
calendar. Although you will be required to collect data for ALL laboratory experiments (as notebooks will be
picked up periodically), you will only be required to turn in formal lab reports for four of the seven laboratories
we perform. A more detailed description of the grading criteria for laboratory notebooks will be given
to you before the first one is due. Your laboratory notebook should demonstrate that you have:
1.
2.
3.
4.
developed concept understanding and skills that have been introduced in the biochemistry laboratory
an ability to share information in written and graphic form
an ability to apply biochemistry content knowledge to real world situations
learned to find information using biochemistry research journals found in most libraries
Each entry in your notebook should include pertinent information about (1) changes to the laboratory
procedures found in the handout, (2) observations, (3) data collected, (4) conclusions, and (5) answers to
study questions, when applicable. You will receive points for complete entries in your lab notebook.
Each FORMAL laboratory report should include the following information:
Abstract - You should describe the point of the current experiment, the experiment performed, and a
summary of the results. The abstract should not be longer than one paragraph.
Materials and Methods – DO NOT reproduce in detail the protocols described in your lab handout, just
briefly describe the stepwise procedure and its purpose. Document any revisions you made to the
protocol, and include observations made during the experiment.
Data - Submit data in written, tabular or graphical form as appropriate. Some laboratories will have
specific data that needs to be recorded, for example, recording of the relative migrations of bands in
DNA or protein gels.
Conclusion – Describe very briefly the end result of your experiment.
Study questions - Address the analysis questions at the end of each laboratory exercise handout. If
these questions cannot be answered due to uninterpretable results, then offer thoughtful explanations
for why results are inconclusive.
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LITERATURE REVIEW
The laboratory experiments you will perform this semester represent some of the standard and
current methods of experimentation in biochemistry labs. A familiarity with the research literature will
increase your understanding of the methodology. Search biochemistry journals for articles published
this year (2009 publish date) that address a scientific question using the methods we will explore
during lab. Since some techniques are addressed over two or more laboratory sessions, below are
the 4 literature review topics, reference to Lehninger book chapter and the dates the reviews and
revisions are due:
Topic 1:
An article that uses at least two physical methods of protein purification (we will use gel filtration
chromatography)
Lehninger: Chapter 3
Review due date:
Revision due date:
September 16, 2009
September 30, 2009
Topic 2:
An article that characterizes an enzyme to determine initial velocity, Vmax and Km
Lehninger: Chapter 6
Review due date:
October 12, 2009
Topic 3:
An article that describes ELISA or Western blotting as a means of identifying the presence of protein
by using this hydridization procedure
Lehninger: Chapter 5
Review due date:
Revision due date:
October 28, 2009
November 11, 2009
Topic 4:
An article that describes the cloning of a DNA molecule(s)
Lehninger: Chapter 9
Review due date:
November 23, 2009
In your review include a copy of the article. Provide a brief 3 page (I will not read past 3 pages!),
double spaced description of the article that includes the hypothesis being tested and the scientific
rationale behind the experiment described (i.e. Why are the experiments important? What disease or
biological process do the experiments examine? Why did the article intrigue you enough to be
chosen?). Some additional reading from broader resources (including textbooks, reference books,
and review publications) is strongly recommended to help address these questions. Often, these
general references are cited in the reference section of the research article that you have chosen. A
more detailed description of the grading criteria for literature reviews will be given to you
before the first one is due. Two literature reviews will be returned to you for revision and
subsequent re-submission. The dates for these events are on the syllabus.
The following are some suggestions of available journals in the Creighton libraries to complete your
literature searches.
Reinert Alumni Library:
Proceedings of the National Academy of Sciences of the USA
Nature
Science
Journal of the American Chemical Society
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Health Sciences Library:
Biochemistry
Proceedings of the National Academy of Sciences of the USA
Nature
Science
New England Journal of Medicine
American Journal of Human Genetics
WEBSITES
Reinert library e-journals site
http://atoz.ebsco.com/home.asp?Id=383
National Library of Medicine database
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi
Elsevier Science Direct database
http://www.sciencedirect.com/
If needed, please see me for hints and assistance in your literature searches and for additional research
journal recommendations.
ELECTRONIC ITEMS IN CLASS
You are expected to turn off all items that emit sounds and noises that may interrupt class (cell phone, pager,
watch alarm, iPod, etc.).
ACADEMIC MISCONDUCT
Please reference the academic honesty procedures on Creighton's website.
http://www2.creighton.edu/fileadmin/user/CCAS/docs/acadhonesty.html
The academic misconduct code includes “representing the work of others to be one’s own (cheating on an
exam, plagiarizing papers, etc.), tampering with the experiments of others, defacing or tampering with library
or student materials or facilitating dishonesty on an exam.” Misconduct also includes using unauthorized
materials in academic exercises, falsification of records, unauthorized possession of examinations,
intimidation, and all other actions that may improperly affect the evaluation of a student’s academic
performance. This includes situations in which you notice or are aware of cheating by someone else and do
not report it immediately. A zero will be recorded for the exam/laboratory report/literature review. Please do
not abuse the trust I have in you.
WEATHER
If the University is closed due to severe weather we will not have class.
LAB SAFETY
A biochemistry laboratory is a potentially unsafe place to work. Some of the chemicals are mutagenic,
carcinogenic, toxic, and/or caustic. Some instruments are potentially hazardous in that they emit ultraviolet
light or high voltage. It is imperative that you wear long pants and closed toe shoes to lab. A laboratory
coat or apron (provided), goggles, and gloves (provided) may also be necessary when entering the lab. All
bacterial strains are potentially hazardous. The Escherichia coli strain used in the lab is weakened and is not
considered pathogenic under normal circumstances. Bacterial waste will be disposed of in a specific manner
and instructions will be given during these laboratory periods.
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GRADING
Formal lab reports
Labs 1, 4, 5
Lab
6
15 points each
25 points
Lab notebook pickup
Midterm check
Final check
20 points
10 points
Quizzes
Midterm
Final
50 points
50 points
Literature Reviews
Topic 1
Topic 2
Topic 3
Topic 4
10 points
15 points
20 points
25 points
TOTAL
300 points
Revisions
10 points
20 points
GRADING SCALE
90.0%-100%
85.0%-89.9%
80.0%-84.9%
75.0%-79.9%
70%-74.9%
60%-69.9%
<60%
A
B+
B
C+
C
D
F
A more detailed description of the grading criteria for lab reports and literature reviews will be given to you
before each of these items is due.
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LABORATORIES
LAB 1 – In order to study the structure and function of a protein, it must be purified using one or several
common techniques. Over two weeks you will purify a protein by gel filtration chromatography, determine its
molecular weight and investigate the importance of protein structure to its function.
------------------------------------------------------------------------------------------LAB 2 - In this lab you will isolate nuclei from calf thymus and then extract the DNA from the nucleus
------------------------------------------------------------------------------------------LAB 3 – In this lab you will use electrophoresis to analyze the hemoglobin protein in normal and mutated
forms.
------------------------------------------------------------------------------------------LAB 4 - In this experiment you will prepare an extract from wheat germ and then determine the initial velocity
(Vo) of the reaction catalyzed by purified acid phosphatase and by the acid phosphatase activity present in
the extract. This will enable you to estimate the amount of the enzyme that is present in the wheat germ.
You will also examine the effects of substrate concentration on the reaction velocity. Data are analyzed using
Michaelis-Menten and Lineweaver-Burk plots to calculate the values of Vmax and Km.
------------------------------------------------------------------------------------------LAB 5 - You will explore Enzyme-Linked ImmunoSorbent Assay, an effective technique to identify and
characterize a specific protein in a complex mixture. The interaction between antigens and antibodies and
the use of chemical tagging is explored in this laboratory.
------------------------------------------------------------------------------------------LAB 6 - The basic strategy following in this series of experiments is one that is used by scientists around the
world to clone DNA molecules from literally thousands of prokaryotic and eukaryotic organisms. You will
digest phage lambda DNA with EcoRI and ligate the resulting DNA fragments to EcoRI-digested plasmid
pUC18 in order to create a series of recombinant plasmids. The recombinant molecules are characterized
by electrophoresis and then introduced into competent E.Coli cells by transformation. The cells are plated
onto nutrient agar containing ampicillin and X-gal. The plasmids from overnight cultures will be isolated by a
unique mini-prep procedure that yields high quality plasmid DNA without the use of toxic organic solvents.
You will also perform restriction digests of your isolated plasmid DNA. You will determine the size of the DNA
fragments you have cloned by agarose gel electrophoresis.
------------------------------------------------------------------------------------------LAB 7 – This crime scene investigator lab involves experimentation used in DNA profiling. The lab introduces
the concepts of the Polymerase Chain Reaction (PCR), which is one of the most powerful techniques used in
biochemistry. The polymerase chain reaction (PCR) is widely used in forensics, diagnostics, paternity testing,
criminal cases, and archeological procedures.
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