BIOLOGY 204/205 Advanced Genetics Laboratory
TABLE OF CONTENTS
Introduction………………………………………………………………………. p. 2
MODULE 1: Recombinant DNA………………………………………………. p. 14
MODULE 2: Gene Expression…………….…………..………………………. p. 28
MODULE 3: Detection of the ALU Insertion; DNA Fingerprinting ……… p. 40
MODULE 4: Proteomics……………………………………………….………. p. 45
MODULE 5: RNA Interference…………………………………………………. p. 53
Appendix A: Solutions Guide……………..……………………………………. p. 63
Appendix B: Sterile Technique…………………………………………………..p. 78
Appendix C: Spread Plate Technique………………………………………... p. 78
Appendix D: Pipette Use……………………..……………………………...... p. 79
Appendix E: Pipette Exercises………………………….……………………...... p. 81
Appendix F: GST Plasmid Map……………………………...……………...... p. 82
Appendix G: DNA/Protein Markers……………………………………...….. p. 83
Appendix H: Streak Plate Method …………………..………………………... p. 84
Appendix I: PCR Reagents and Conditions for 1.17………………………… p. 85
Appendix J: Pierce Protein Assay for Module 2 ……………………...…….. p. 86
Appendix K: Protein Gel Set up………………………………………………. p. 87
Appendix L: Pierce Protein Assay for Module 4 …………………………….. p.88
BIOLOGY 204/5
Advanced Genetics Laboratory I and II
--- Introduction ---
Module 1 Recombinant DNA/Bacterial Transformation
This module gives you some of the experience you would receive if you were to sub-clone a gene as a part of your research. That is, once you transform a bacterial line with the plasmid that you isolate, you will need to demonstrate that you have made the transfer of the correct gene.
Goals:
1. To purify a plasmid and transform E. coli with the plasmid.
2. To demonstrate that the transformants carry the plasmid by characterizing the transformants’ phenotypes.
3. Analyzing the size of the DNA plasmid in a cracking gel.
4. Hybridization with the original plasmid in a Southern blot.
5. Amplify the gene inserted into the plasmid by PCR.
6. Sequence part of the plasmid.
Module 2 Gene Expression
This module allows you to determine whether a cell is expressing a gene of interest, either GFP or GST. You will run the bacterial lysate on gels, stain with Coomassie blue to look for a protein of the correct size and perform a Western blot to determine whether the protein of interest was expressed.
Goals:
1. Confirm, using PCR, that the plasmid from the transformed bacteria (from module 1) has the GST gene.
2. Show that bacteria with the gene for GST in the expression plasmid are expressing GST using gel electrophoresis of the bacterial lysate.
3. Show that GST is expressed by the bacteria using a Western blot of the lysate.
Module 3 Human Genetics: Detection of the ALU Insertion; DNA Fingerprinting
This module allows you to carry out processes and procedures that a forensic scientist might carry out.
Goals:
1. Isolation of your cheek cell DNA for polymerase chain reaction amplification.
2. Analysis of PCR products by agarose gel electrophoresis for the Alu insertions in chromosome 8 and a polymorphism called a variable number tandem repeats (VNTR) D1S80 from the noncoding region of chromosome 1.
3. Use the techniques you learned to help solve a “crime” by comparing DNA “found” as evidence, against the DNA of the “victim” and the “suspects.”
Module 4 Proteomics
In this module you will compare the proteins found in wild type and mutant paramecia cilia. You will run a one dimensional polyacrylamide gel, cut out bands of interest, and compare the proteins present in those bands using mass spec.
Goals:
1. Conduct a Pierce Protein Assay to determine the concentration of cilia proteins in samples provided to you.
2. Using SDS-PAGE, run a one dimensional gel and stain with coomassie blue to visualize differences in protein content of cilia types.
3. Cut out bands of interest from gel and prepare samples for mass spectrometry by trypsinizing proteins
4. Analyze results from mass spec
Module 5 RNA Interference
In this module, a specific target gene product of Paramecium tetraurelia is depleted using an RNAi feeding method. You will isolate RNA from the paramecia and determine if there has been down regulation of the gene product in the RNAi treated population compared to the control.
Goals:
1. Isolate RNA from harvested paramecia cells
2. Synthesize cDNA from the mRNA collected
3. Determine the level of target endogenous mRNA by semi-quantitative Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR)
Biology 204/205 Advanced Genetics Laboratory
Grading Policy
Biology 204 and Bio 205 are four credit courses. You will complete Modules 1 & 2 and a grant writing exercise during the fall semester for Bio 204. For Bio 205 in the spring semester, you will complete two modules (Modules 3, 4 if you have already completed 1 and 2) and Module 5.
The format of the course is a short introduction and two formal meeting times per week. The emphasis of the course is on experimental design, techniques, data gathering and analysis. Work at the bench is given priority over work in a lecture setting. The modules are designed to approach real situations in ongoing research projects. Therefore, the modules are not necessarily designed to be finished in three hours. A few labs will run long, taking 4-5 hours to finish. A few labs will be relatively short. Students will sometimes need to return on an alternate day, usually at their own convenience, to perform a short manipulation. Sometimes an experiment does not work and it has to be repeated. Coming to class well prepared and following directions carefully will cut down on potential mistakes!
Grading:
Your grade will be based on the following components, each with approximately equal weight:
1. Performance in laboratory
2. Discussion of experiments in class
3. Notebook (these will be checked weekly)
4. Laboratory report
5. Grant proposal (Bio 204) or Lab meeting presentations (Bio 205)
6. Final oral exam covering both lecture and laboratory material
Supplies:
You will need the following:
· Laboratory manual
· Bound laboratory notebook
· UV safety glasses
· Sharpie permanent marker
· Transparent tape
Note: It is important to read over the procedures in the laboratory manual and the corresponding information in the Molecular Biology Laboratory Atlas before coming to class. Be prepared to start work after an introduction by the instructor or TA. Check the laboratory calendar so that you know when each module will be done.
Laboratory Notebook Guidelines
· Bound notebook; no loose-leaf
· Record in blue or black ink
· Number all pages
· Date all entries
· Name, course number and email address should be on front cover
· Reserve 3 pages at the beginning for the table of contents; keep up to date
· Mistakes should be crossed out with a single line through the entry then initialed
· Do not skip pages, do not rip pages out
· Unused portions of a page should have a diagonal line drawn through the blank portion
· Each experiment should begin on a new page
· All data, calculations and graphs should be entered directly into the notebook
· Neat, orderly, complete
Your notebook should provide enough detail so that another Advanced Genetics student could pick it up and repeat your procedure by following your entries. You should include all of the following information: What was done and why, who suggested it, who did it and when it was done, what results were obtained and what conclusions were drawn.
NOTE: NOTEBOOKS WILL BE CHECKED WEEKLY AND GRADED ACCORDINGLY!
Laboratory Report Guidelines
You will be asked to write a formal report of one of the results from one of the lab modules. You will prepare this report as you would prepare a manuscript for publication, with introduction, methods, results, and discussion sections. To aid your preparation of this report, you should go to the library early in the semester and find a short article from Genetics from the last 5 years (download a pdf version or photocopy from a paper journal, the library has both formats). The format in Genetics is appropriate for your report. Below is a description of the content and length of each section.
The report in its entirety should not exceed 10 pages in length. It should be printed double-spaced, with no less than 1-inch margins. It must be in 12-point size in a common font. Each section except the introduction should be started by its section name, in bold type. At the head of the report, you should provide a title that indicates which exercise you are writing about and your name. Whenever possible, you should strive to write succinctly and in the active voice.
Abstract: 250 words summarizing the experiment.
Introduction: The introduction provides an overview of what the report is about, including why the exercise was done (the goal of the exercise) and an explicit statement of the hypothesis or hypotheses being tested. Background information about the biology underlying the exercise should be included in the introduction. Recommended length: 1.5 pages.
Methods: The methods section must be detailed enough to allow the reader to repeat the exercise. You do not need to repeat the detailed description of the protocols in the laboratory manual, but you should refer to the methods in the manual (Format: Laboratory manual Page x-y) at the appropriate points. Recommended length: 3 pages.
Results: The results section reports upon what happened during the exercise. You must include photocopies of the final gels and provide in tabular form other measurements and data you collected. Each figure should have a brief descriptive caption, and each table should have a title. However, it is not sufficient to simply insert these figures and tables. You must interpret your results in the text of the section, with references to the appropriate figure or table (Format: Fig. 1, Table 2A). Recommended length: 2 pages.
Discussion: In the discussion, you should briefly re-introduce the main goal or hypothesis presented in the introduction, and then describe how your results are related to the goal or hypothesis. In subsequent paragraphs, you should discuss any failures to obtain results, and describe what you believe happened and what you would do differently to correct each problem. This is your opportunity to show how well you understand the molecular processes underlying the protocols! Recommended length: 2 pages.
Grant Writing Guidelines
The grant proposal must be based upon a novel concept that could be explored within the technological and financial limitations of our laboratory, using model organisms only. These limitations will aid you in narrowing your choice of topic.
A. Topic: The topic chosen should be novel, which means that no one else has worked on this same exact problem before. However it should also be one that can realistically attacked in the context of this laboratory. You should be able to put this topic into a broader context; Why is this an interesting problem? What has already been done with this problem in the past?
1. From this topic you must develop a testable hypothesis. This means you can develop an experiment that will result in data that leads you to clearly be able to reject or accept the hypothesis.
2. The experiment(s) will utilize techniques that you can do within this laboratory or with our collaborators.
3. If you are currently working in a laboratory outside of this class the topic you choose MUST be independent of that laboratory. You are not permitted to work on the same model organism or a topic that is related to your outside research.
4. You are permitted, indeed encouraged, to discuss possible topics with classmates, friends, family and other faculty.
B. Model Organisms: Your experiments must be limited to classic model organisms from molecular genetics that are readily available, easily maintained and for which we have appropriate technologies to care for and manipulate. Examples include: Drosophila, bacteria, Paramecium, C. elegans, yeast, small plants like grasses. You cannot work with organisms that are difficult and expensive to maintain (i.e. mice or other small rodents, fish, large vertebrates, humans, etc.)
C. Grant Format: Below is the format that your grant must follow. All text must be double spaced 12 point type with 1 inch margins. Make sure your grant contains all of the information within the guidelines given:
1. Cover Page - Fill out the cover page provided completely
2. Table of Contents - Page two is a table of contents. Provide the page number of each category. Number pages consecutively at the bottom right of each page throughout the application (including the cover page as page one).
3. Biographical sketch - A one page biographical sketch of the Principle Investigator (PI; this is you!). This contains your name, your date of birth, your education history, your previous relevant employment and a brief description of your prior experience that allows the reviewer to recognize you are capable of doing the proposed research.
4. Research Plan - This section should be 10 pages total. It should contain sufficient information needed to evaluate the project, independent of any other documentation. Be specific and informative, avoid redundancies. All tables, graphs, figures, diagrams and charts must be included within the 10 page limit. The following questions must be addressed in your research plan:
a. What topic will you be trying to address?
b. Why is this research important?
c. What has already been done in this field/topic?
d. What exact experiments will you be performing?
e. What are your expected results? (This is to clearly show your hypothesis. What results will allow you to accept your hypothesis? What results will cause you to reject your hypothesis?)
5. Literature Cited - All references cited in the proposal (of which there should be many) must be listed alphabetically by first author. They must include all authors, year of publication, complete title of article, journal name (no abbreviations or websites), volume and page numbers.
6. Appendix - In an appendix not to exceed 4 pages you may provide detailed descriptions of your protocols, with all appropriate references in your citations*. Your descriptions of the protocols should be as detailed as those provided in the laboratory manual.
*Protocols: If you are using protocols in the laboratory manual you may cite the manual itself as a reference. If, however, you propose other protocols you find and cite the original reference that describes the protocol.