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2005 HSTA Summer Institute Inquiry Experience: |
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People in the lab that will work with HSTA teachers and students
Dr. Laura Gibson (PI)
Jim Fortney (Research Assistant)
Heather O'Leary (Doctoral Student)
Suzanne Davis (Microbiology Doctoral Student)Where are we? Room 2323 in the Health Science Center
Goals for the week…
- Become comfortable with using new lab equipment that may not be familiar to you; what are other potential uses of the equipment that we use?
- Identify ways that DNA can be expanded…how can we make enough of it to study? Why would we want to make more?
- Understand how we can look at a DNA fingerprint; how is it different from a typical fingerprint? Which is more useful?
- Identify how bacteria can be useful to us in the lab; what are some questions that bacteria could help us answer other than those included in the exercise that will be part of your HSTA week?
- Think of a question and design an experiment that you would do if this lab was yours…
- Have fun!
The week’s activities will be divided into blocks which are described below. For each of the procedures, you will be provided a detailed description in writing and you will get “hands-on” help as well. The blocks of activities are not listed with specific times because we will move at the pace that is best. However, the experiments have been designed so they will fit into the time you have been allotted for the inquiry experience.
Block I- Introduction
We will start with an introduction to the lab and the people that you will work with; everyone is here to answer your questions, so just ask! The lab rules below are primarily to make sure everyone is safe, and that we are compliant with University regulations. In addition, they make sure that the atmosphere is one in which everyone is comfortable and can have a good experience. All of them are equally important….
Lab rules include:
- no food or drink
- must wear shoes at all times
- appropriate language
- respectful and considerate behavior
- wear safety glasses when using the UV light box
Block II- Transfection
During this block we will transfect a plasmid (circular) DNA into bacteria. We will use chemicals to make the bacteria receptive to the DNA, then grow the bacteria on plates that have been coated with media (agar) that has been optimized for bacteria expansion. This will allow us to make many copies of the plasmid DNA as it is copied and passed on to new bacteria as they divide. As the bacteria divide, they will form little clumps (colonies) on the layers of agar. We will be able to see these colonies, and confirm the presence of the plasmid as described below in Block IV .
The general steps of this block are summarized below:
Treat bacterial cells with calcium chloride to enhance uptake of plasmid
Incubate the cells with plasmid DNA
Transfer the bacteria onto agar plates that contain ampicillin (so only the bacteria with the plasmid will grow…and not bacteria from our hands, etc.)
Colonies should appear in 2-3 days…
When would expanding DNA be useful?
Why would we choose to use bacteria as a tool for this exercise?
What are other ways to expand (make copies of) DNA?
Block III- Cloning plasmid DNA into bacteria ednesday
During block II we learned to transfect a plasmid into bacteria. We will now use this procedure again to put another plasmid into E. coli bacteria. This time we will grow the bacteria in liquid media (instead of on agar plates). The bacteria will grow overnight, and we will use the bacteria containing plasmids during Block IV.
The general steps of this block of activity are summarized below:
- Treat bacterial cells with calcium chloride to enhance uptake of plasmid
- Transfer some of our plasmid DNA to the “competent” bacterial cells
- Add nutrient broth to the tube of bacteria + DNA and incubate for 30 minutes
- Plate the mixture on agar plates
- Allow the bacteria to grow overnight…in addition to your colonies that will grow, we will grow up some “bulk culture” that will provide enough bacteria for everyone to isolate plasmid DNA in Block IV.
Block IV – Isolation and analysis of plasmid DNA
Before we get started with main part of this block we will check for colonies from our Block II transfection; anything glowing yet? We will look at our colonies on a UV light box as mentioned above. WEAR GLASSES AT ALL TIMES! Any colonies (groups of bacteria) that have the plasmid in them will glow in the dark because we have transfected in a luciferase gene. This will only take us a few minutes and then we will begin the main part of this block. If nothing shows up, we will let the bacteria grow another day…
We will spend most of the time during this block learning to isolate our plasmid from E.coli. Then we will cut (digest) the plasmid DNA and separate by gel electrophoresis. Restriction enzymes are unique enzymes that recognize DNA at specific sequences and cut it. After it is cut into smaller fragments, we can separate it by size on agarose gels. We stain the gels so we can visually detect the DNA patterns. These can be photographed for permanent records.
General steps are outlined below (remember…you will get detailed protocols and instruction when you come to the lab):
- We will divide up the culture that we grew overnight; this contains the same bacteria and plasmid as the plates that you established
- Centrifuge to pellet the bacteria
- Lyse the pellet to release the plasmid DNA
- Use isopropanol to precipitate the DNA after some “clean up” steps…this sample will contain our plasmids (circular) DNA
- Restrict (cut up) the DNA using Eco RI , a restriction enzyme
- While the DNA is being restricted, we will pour agarose gels
- Separate our DNA on agarose gel…stain and take pictures
Check our colonies again if they were not detectable earlier.
During this exercise you will use restriction enzymes to distinguish between two different species of DNA. We do not use any human DNA, so there are no biohazard concerns.
General steps include:
- Cutting the DNA with two different restriction enzymes (DNA will be provided as unknown #1 and unknown #2)
- Separate the fragments by gel electrophoresis
- Stain gel and photograph
What are the differences in the DNA patterns?
What might explain these differences?
Block VI – Experimental Design and Graphing
Estimating size of DNA band based on molecular weight marker standards
Organize data and discuss interpretation of all the results
Design an experiment to answer a question of your own…Be creative!!!
Developed on July 11, 2005 by Sohail Khan