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Purdue University
Botany & Plant Pathology
BTNY 11000
Peter Goldsbrough

10-1 FALL 2012 NAME_______________________ BTNY 110 LAB SECTION________________ LABORATORY #10 (MOLECULAR BIOLOGY AND BIOTECHNOLOGY) Lab #10 Objectives: 1) learn about restriction enzymes and what they do 2) become familiar with gel electrophore sis, how it works and how to interpret the results 3) know what the term “genetically engineered/modified” means and observe a real-life example used across the US WHAT IS GENETIC ENGINEERING? Genetic engineering is the process of altering the genetimaterial of an organism or introducing new genetic material into the organism. Normally this is done in order to introduce a desirable or beneficial characteristic into the organism. Genetic modification through traditional breeding methods has been acco mplished throughout the history of humankind in the domestication of plants and animals. For example, a plant with a beneficial characteristic in terms of height might be crossed with a plant with a beneficial characteristic in terms of fruit flavor. Both beneficial traits end up in some of the offspring and these are selected and self-fertilized until the traits breed true. However, this traditional breeding approach can take many years to accomplish and is limited to transfer of genetic material between very closely related plants. In very recent years scientists have learned how to manipulate genetic material in the laboratory and to move genes between organisms that may be distantly related. For example, genes from animals, or even bacteria, can be introduced into plants. These methods are now in common use in molecular biology laboratories all over the world. Genetic engineering using molecular techniques involves several distinct steps: 1. The first step is to identify the gene with desirable characteristics. Let’s say, for example, that we want a gene in soybeans that would cause the crop to be resistant to a herbicide like glyphosate. We might try to grow bacteria in a medium containing glyphosate. If we can find a bacterium that can grow in the presence of glyphosate, we would say it is resistant to this herbicide. 2. The next step would be to isolate the gene responsible for the resistance and try to make many exact copies of it ( cloning). This can be a very difficult task, but it is often possible. As you know, a gene is made up of a linear chain of nucleotides. The sequence of nucleotides in a specific gene is unique, corresponding to the protein encoded by the gene. Using molecular tools of cloning, the gene of interest can be is olated and multiplied in bacterial cells. Very often the gene is inserted into a very small circular chromosome called a plasmid that can be reproduced in the bacteria. 10-2 3. Once the gene is cloned into a plasmid, it can also be introduced into plant or animal cells. This process is called transformation. There are many different methods used to transform cells or plants. Two of the most common methods include using a gene gun (biolistics) and using a bacterium to infect the cells and transfer the pl asmid into the cells. 4. Transformed cells can then be selected and grown in tissue culture to form entire new plants that can reproduce sexually and carry the new gene on generation after generation. This process is called regeneration. The scenario described above has actually been carried out for a glyphosate -resistance gene to produce Round-up Ready Soybean. Likewise, plants have been transformed with many other genes that lend desirable characteristics. TODAY’S LAB EXERCISE: In lab today you will have an opportunity to use a very common and important method in molecular biology. You will be given two samples of bacterial plasmids. Both samples contain the same plasmid, into which a foreign gene has been inserted. One of the samples has been treated with an enzyme used to cut the foreign gene out of the plasmid. The other sample is untreated. The figure on the next page illustrates how the gene was placed in the plasmid, and suggests how it could be removed. To introduce a foreign gen e into a plasmid, both the plasmid and the gene (from another source) are cut with restriction enzymes to produce complementary sticky ends. A restriction enzyme (pictured in the figure as a pair of scissors) is an enzyme that recognizes a specific DNA sequence of 4 to 8 nucleotides and cuts the DNA in a precise manner at that site. There are many known restriction enzymes that identify and cut at different DNA sequences. The cut ends of the plasmid and the foreign gene bind each other and are attached to gether by an enzyme known as ligase (pictured as glue). Once a foreign gene has been incorporated into a plasmid, as illustrated, it can also be cut out of the plasmid. The gene is released from the plasmid by cutting the DNA with specific restriction enzymes. The resulting pieces of DNA (cut plasmid and gene) can be visualized using the technique of gel electrophoresis. 10-3 Figure 18.8 from PLANT BIOLOGY by Graham, Graham, and Wilcox. 2003. Pearson Education Inc. 10-4 In lab today we will perform agarose gel electrophoresis to allow us to visualize pieces of DNA, including an intact plasmid, as well as a plasmid containing an inserted gene which has been released by cutting with a restriction enzyme. Electrophoresis separates DN A based upon its size, or number of nucleotides. The gel is composed of solidified agarose. The DNA will pass through the spaces left by the agarose molecules within this gel matrix. The force that drives the DNA through the gel is electricity that flows from the negative electrode to the positive electrode. The negatively charged nucleotides of DNA cause it to migrate toward the positive electrode and away from the negative electrode. The speed with which any one piece of DNA migrates through the gel is dependent upon its size. Smaller pieces of DNA move more rapidly and easily through the gel spaces while larger DNA fragments get hung up and move more slowly through the gel. The gel also contains a fluorescent dye (ethidium bromide) that binds to DN A. So, when you are finished with the electrophoresis, you will be able to observe the DNA as fluorescent spots or bands under an ultraviolet lamp. PROCEDURES Your TAs will prepare an agarose gel for each group. Numbers 1 -6 below describe the procedure your TA followed in preparing the agarose gels. Each group should pick -up a gel from the hood and start with #7 under “Loading your gel.” ( Note: Ethidium bromide is a carcinogen. Wear gloves whenever you handle the solution or gel.) Preparation of an agarose gel: 1. Add 50ml of 1X TAE buffer to a 125mL Erlenmeyer flask. 2. Weigh out 0.5 grams of agarose and add it to the flask. 3. Microwave the flask on high until the agarose dissolves (1 -2 minutes). Do not let the solution boiling out of the flask. Check your gel casting tray and comb to ensure that it is set up and ready for you to pour your gel. Put on gloves before the next step. 4. Carefully remove the hot flask and add 2.5 µL of ethidium bromide. 5. Place the flask in an ice water bucket and gently swirl for 25 seconds. 6. Pour the gel solution into your casting tray and allow it to solidify (20 -30 minutes). Loading your gel: 7. Gently remove the gel comb. 8. Remove the gel from the casting tray and place it in the gel box so that the gel is submersed in buffer. 10-5 9. Check the contents of your DNA samples. If they have been splashed onto the sides of the tube, use a microcentrifuge to pull everything to the bottom of the tube. 10. With a P-20 Pipetman load 20 µL of each D NA sample with dye into a separate well. Be sure to keep track of which sample you loaded into each well. (Note: The setting on the pipetman should read 200 from top to bottom) 11. With a P-20 Pipetman load 20 µL of DNA marker into a lane adjacent to one of your sample lanes. The DNA marker provides
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