Return to Biology/Microbiology Home DirectoryThis lab provides students with experience in several technologies used
in molecular biology. Students will express a recombinant human gene in
insect cells and perform assays on the purified protein. In a second exercise,
the polymerase chain reaction (PCR) is used forensically to differentiate
eggs from different species of fish. Finally, site-directed mutagenesis
is used to target a cytoplasmic protein to the nucleus of yeast.
We will meet Mondays from 1:10 to 4:15 in room 316 Cowley Hall. Occasionally students will have to come in at other times to check on cell cultures or harvest bacterial cultures. These times are indicated in italics. Concurrent enrollment in BIO 435/535 is required. Lab coats and glasses are also required.
All of the links to programs used for DNA sequence analysis and protein modeling are found in UW-GenWeb.
Day before:
start overnight cultures of pGEM-AT and pVL
2. Plasmid minipreps. Restriction digestions. Prepare competent
cells.
Homework
#1 due (25 pts)
3. Gel purification. Ligation. Test transformation.
Day after:
put ligations and plates in refrigerator
Introduction
due (25 pts)
4. Transform ligations. Calculation of transformation efficiency.
DNA sequence analysis (open reading frames
and restriction mapping).
Day after:
put plates in refrigerator
6. Measure DNA concentrations, homologous recombination, tissue
culture. Molecular modeling of proteins.
3 days later:
harvest virus. Amplify by infecting new cells.
Quiz
#1 (50 pts)
7. Harvest media, affinity chromatography, immunoblot, ELISA,
dialysis
Day after:
collect proteins from dialysis
9. PCR of a paddlefish and sturgeon DNA. DNA sequence analysis
(alignments)
10. RFLP analysis of the PCR product. DNA sequence analysis
(primer design)
Rough
draft of paper due
11. Site-directed mutagenesis.
Quiz
#2 (50 pts)
12. Transformation of yeast
Day after:
put plates in refrigerator
14. Catch up day or lab final (100 points).
Labs: Mondays from 1:10 to 4:15 in room 316 Cowley
Hall.
Exams: (100 points) There will be one lab final.
Homework and Quizzes: (150 points) There will be
problem sets and quizzes given in lab stressing calculations commonly used
in molecular biology.
Research Paper: (150 points; 100 points for final paper, 25 for rough draft, 25 for review). Each student will write research paper on the results from their research project (maximum 6 pages). The paper will be in the format of the Journal of Biological Chemistry. Complete with Abstract, Introduction, Materials and Methods, Results, Discussion. Figures will be attached to the end of the paper, after the References section. This paper will contain the data from the bulk of the work done in this lab. Prior to final submission you will exchange papers with other students for "peer-review". This is a very important step in getting a paper published. Other scientists review your work and make suggestions and comments. You will then respond to these comments by making some or all of the suggested changes in your paper.
Article for a newspaper: (25 points) In the last
lab we will be using techniques employed in molecular forensics to determine
the source of a DNA sample. You will write an article describing the results
of this important test. You can embellish the story any way you wish, a
murder, paternity case, poaching etc. Somewhere in this article you should
include a paragraph explaining the technique [PCR and RFLP mapping] in
a way that a non-scientist can understand what you did.
Lab notebook: (25 points) Your lab notebooks will be collected and graded at the end of the semester.
Total points: 450
Grading
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