BIL 256 - Cell and Molecular Biology
Lab
Class Web Page: http://www.bio.miami.edu/courses.html
|
Dan DiResta |
Tanya Hawley |
|
165 Cox Science |
208 A Cox Science |
|
284-2180 |
284-6420 |
Cell and Molecular Biology Lab consists
of a 1 hour class discussion and a 3 hour lab exercise. The purpose of the
discussion is to plan lab experiments. You will be given information on a
particular experimental question and asked to design an experimental protocol
to best answer the question. This is not a "cook book" lab. The
philosophy behind this lab is to involve you in the process of how science is
done. By the time you come into the lab, you should know exactly how you will
carry out the experiment as well as why you are doing it. Many of the
techniques you will use require a level of technical competence that is only
achieved through practice and experience. Therefore, carrying out a successful
experiment is not as important to your grade as understanding the limitations
of your experimental technique and your intelligent analysis of experimental
results.
Much of the data analysis will involve
the use of statistical and graphing computer programs. You will be required to
calculate means and standard deviations of repeated measurements and test
whether the difference between means is significant. You will use linear
regression analysis to calculate a linear equation that best fits your data.
Familiarity with spreadsheet programs such as EXCEL will be helpful in
analyzing and writing up experimental results.
During the lab you will be working in
pairs. It is expected that each person will contribute equally to planning the
experiment and collecting and analyzing the data. You will be required to keep
an up-to-date lab notebook and record all appropriate experimental details.
Experimental write-ups, using standard scientific format, will be due the
following week. Grades will be based on participation in experimental
design and planning, understanding of the experimental system, analysis
and write-up of experimental data, and notebooks.
|
Week |
Experiment |
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|
1/22 |
Spectrophotometry
- Demonstration of Beer-Lambert Law
using the dye dichlorophenol-indophenol (DPIP).
Determination of the absorbance spectrum of DPIP. Constructing a standard
curve. Determination of an unknown concentration of DPIP. |
|
1/29 |
Protein Assay - Estimation of protein concentration in an unknown
solution using Biuret and Bradford techniques. Constructing a protein
standard curve. Linear regression analysis. Calculation of protein
concentration. |
|
2/5-2/26 |
Enzyme Kinetics - |
|
3/5 |
Electrophoretic Analysis of Native Proteins 1. Separation of
normal hemoglobin (HbA) and sickle hemoglobin (HbS), the
most common variant causing sickle cell anemia. Comparison of the electrophoretic patterns of two proteins differing in
only one amino acid in the beta chain of the molecule. |
|
3/19 |
Electrophoretic Analysis of Native Proteins 2. Comparison of lactate dehydrogenase
isozymes. LDH separated electrophoretically
and visualized by activity staining. |
|
3/26 |
Molecular Weight
Determination of Denatured Proteins
- SDS-polyacrylamide
gel electrophoresis of protein standards and two unknown proteins to
determine molecular weight. |
|
4/2 |
Affinity
Chromatography - Isolation and
purification of horse serum albumin by affinity chromatography and SDS-PAGE. |
|
4/9 |
Western Blot- Serum proteins from 5 different species are electrophoresed, transferred to nitrocellulose membrane and assayed for crossreactivity with human serum proteins. |
|
4/16 |
ELISA Immunoassay - Specificity of antibody-antigen interactions using
serum proteins from several species and anti-rabbit IgG
antibody. |
|
4/23 |
Notebooks
Due |