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Lab Exercise Outline
As described in Module 3 - Home, the polymerase chain reaction (PCR) is one of the most commonly used methods for cloning genes. PCR can also be used to determine whether a gene or gene fragment is present in your bacterium. In this lab exercise, you will use PCR to determine whether a gene conferring antibiotic resistance is present in the bacteria you worked with in the Module 2 - Lab Exercise

Experimental objective Study question Methods Setting up the reaction Preparing the template Assembling the PCR Thermocycler programming Preparation of agarose gel Loading of agarose gel Staining and photographing agarose gel Results Analysis lab exercises for print-out

Experimental Objective In Module 2 - Lab Exercise, you allowed two species of bacteria to exchange genetic information. This exchange allowed one, or possibly both, of the bacteria to grow on a combination of antibiotics that neither one of them could grow on before. What we do not know, though, is which genes for antibiotic resistance were passed to which bacteria. To solve this mystery, we are going to use the Polymerase Chain Reaction (PCR). PCR allows the amplification of specific pieces of DNA, and is used medically to differentiate between species of bacteria. In this experiment, we are going to use four different sets of Primers. Their names and the pieces of DNA they are specific to are listed below: Primer Set Name Region of DNA amplified Primer Set A Amplifies the gene that gives resistance to Antibiotic A Primer Set B Amplifies the gene that gives resistance to Antibiotic B Primer Set C Amplifies a gene that is only found in Pseudomonas Primer Set D Amplifies a gene that is only found in E. coli Study Question: If you preformed a PCR reaction with each of these primer sets on the E. coli strain that you started with in Module 2 - Lab Exercise, which primer sets would you expect to amplify DNA? Methods: Setting up the reaction: There are several important components of a PCR reaction: Template This is the DNA sequence being amplified. In your reaction, this sequence is either on the genome or on a plasmid. Primers Small pieces (~20 bp) of single stranded DNA that base-pair with the 5' ends of the strands being amplified. DNA Polymerase This is the enzyme that joins dNTP's together to form a DNA strand. The enzyme used is PCR was isolated from the thermophilic bacteria Thermus aquaticus. Deoxynucleotide-triphosphates (dNTP's) The fuel for the reaction. There are four types of dNTP's, dATP, dGTP, dCTP, and dTTP. dNTP's are joined together by the DNA polymerase to make a new DNA chain. Buffer This is a mixture of salts and other ions that keeps the PCR at the right pH for the DNA polymerase to be active.               Preparing the Template The first component that your group will need for your PCR is the template DNA. Initially the template DNA is inside the bacterial cells growing on the plates. In order to do the PCR, we will need to get the template DNA out of the bacterial cells. 1. Obtain a centrifuge tube with 1 ml of Chelex solution from your instructor. 2. Pick a small glob of bacterial cells (around 1 cm across) off of your plate of media. 3. Add this gob of cells to 1 ml of Chelex solution. 4. Incubate the Chelex/cell mixture in a boiling water bath for 10 minuets. 5. After the incubation, allow the Chelex/cell mixture to cool to room temperature. 6. Centrifuge your Chelex/cell mixture for 1 minute. The Chelix pellet is very delicate, so be careful not to disturb it when removing the your tube from the centrifuge. 7. Place your tube on ice and move on to preparing your PCR reactions. Assembling the PCR Now that the bacterial cells have been broken down, releasing the template DNA, you can begin assembling the PCR reaction. Since there are four different regions of DNA we would like to amplify, we will need to run four different reactions, one reaction for each set of primes. 1. Obtain four 200 µl tubes that contain PCR "beads" and one tube of each primer set from your instructor. The PCR "beads" actually contain several of the components needed in the reaction, dried into a small "bead". The PCR bead contains: The DNA polymerase The dNTP's The buffer 2. Label each of your 200 µl tubes with the name of one of the primer sets. 3. Add 20 µl of the appropriate primer set mix to each 200 µl tube. 4. Add 5 µl the Chelex/cell mix to each 200 µl tube. Since the Chelex will inhibit the PCR, it is important that you take care not to resuspend the Chelex beads and that you take 5 µl of solution from the top of the Chelex/cell mixture. 5. The PCR is now ready to go into the thermocycler. Make sure you have labeled your 200 µl tubes with your group's initials, then bring them to your instructor. Your instructor will place your reaction tubes in the thermocycler and run it using the following program. Thermocycler program 1. Melting Temperature: 95?C, 30 Seconds This step separates the strands of the template DNA 2. Annealing Temperature: 65?C, 30 Seconds This step allows the primer to bind to the template DNA 3. Extension Temperature: 72?C, 60 Seconds This step allows the DNA polymerase to add dNTPs to the primer The thermocycler will cycle through these 3 steps 30 times. The PCR should run for 2-3 hours. Once the thermocycler is done, it will be necessary to run the DNA fragments that you have made out on an agarose gel. Preparation of Agarose Gel The group of students that is to prepare the 1X TBE buffer will need to add 200 ml of distilled water to the bottle of TBE powder (USB #70454). Mix thoroughly. This is now a 10X concentration of the TBE buffer. Pour the contents of the bottle into a 2-liter beaker or Erlenmeyer flask. Carefully add 1800 ml of distilled water and mix until fully dissolved. This is enough buffer for four gels. If only 1 liter of 1X TBE is to be prepared, then use 100 ml of the 10X concentrate and 900 ml of distilled water. Share the extra buffer with another group of students. Each group will need to prepare a gel. Each pair of students in the Investigatory group will load their samples on to this gel. a. Weigh out 1.3 grams of agarose and add it to 100 ml of 1X TBE buffer in a 250-ml Erlenmeyer flask. You can prepare more than one gel at a time by scaling up the recipe (2.6 grams in 200 ml, etc.). b. Place a magnetic stir bar into the flask and heat the flask on a stirring hot plate until the agarose particles are fully dissolved. The solution will have to boil for several minutes for this to occur. If a magnetic hot plate isn't available, heat the materials in a 250 ml or 500 ml beaker and stir frequently. (A microwave oven can be used for this purpose if it is available. If a microwave is used, microwave on high for 1 minute, then swirl the flasks. If the agarose is not completely dissolved, microwave for a few seconds longer until it is.) c. Once the agarose is fully dissolved remove the flask from the hotplate, insert a thermometer, and allow the solution to cool to 70°C. a. While the agarose is cooling, the next step it to prepare the casting tray. At the moment, CIBT has two types of gel apparatus in our lending library, ones that have not gasket on the casting trays and those that do. Examine your casting tray. - If the casting tray has no gasket, place tape across the end of the tray so that it blocks off the open ends of the tray. Place the taped tray on a flat surface (not in the running apparatus). - If the casting tray has a gasket, place it in the gel running apparatus so that the open ends of the casting tray are facing the sides of the gel running apparatus. The gaskets should make a seal with the sides of the gel box. b. If you are using the GelStar® DNA stain, add 10 µl of GelStar® to 100 ml of the cooled agarose before pouring the gel. Wear gloves when handling the GelStar® stain. c. Pour the cooled solution into the tray to a height of 0.8 to 1 cm (approximate). It may be necessary to tip the tray gently to spread the agarose across the entire surface. d. After the gel is poured, place the comb so that it fits in the slots that are closest to the end of the gel-casting tray. (There are two sets of slots in the gel casting tray, one close to the end of the tray and one in the middle of the tray. Do not use the slots in the middle of the tray.) The wells should be positioned closest to the black end (negative) of the running chamber. e. Leave the gel undisturbed for 20 minutes until the agarose becomes firm and opaque. a. When the gel has cooled, remove the gel casting gates from the ends of the tray. b. Add 800 ml of 1x TBE buffer to the gel chamber. The gel should be fully submerged by about 2 - 3 mm of buffer. c. Remove the comb by gently pulling it straight upward. You will load your samples into the slots created by the comb. Loading of the Gel Each group of students will load 4 of the 14 wells in the gel. In addition to your up-to-six samples, your teacher will also give you a sample of DNA size standard. This size standard is actually the genome of a virus, bacteriophage (lambda) that has been previously digested with Hind III into fragments of known length. Remove the restriction digests from the incubator. Use a P20 micropipette to add 5 µl 5X loading dye to each sample. Place all six of the tubes into the microcentrifuge and spin for 5 seconds to make sure that all the liquid is combined in the bottom of the tube. Before you start loading, discuss the order you would like to load your samples in with the rest of your group. It is important that you do this before you start loading your gel, since if you take too long when loading the gel your samples will diffuse out of their slots. It also is helpful if one group loads their standard DNA is loaded in one of the middle lanes and the other loads it in one of the outside lanes. Load 10 µl of each sample into the wells of the gel in the order described below. The loading dye will make your samples dense so they will sink to the bottom of the slots in the agarose gel. Arrange your samples on the gel in the following order (label the wells on the diagram on next page): Place the cover on the apparatus and connect the leads to the power supply. Electrophorese at 185-199for ~90 minutes or until the blue dye (from the loading dye) is near the bottom of the gel. Gels can be run overnight at lower voltage. (Try 35volts for 18 hours if two gel boxes are connected to one power supply.) Staining and Photographing the Gel (if using Carolina Blu™ DNA stain) Gently transfer the gel to a staining tray and cover with Carolina BluTM Stain (250 ml). Leave the gel in the stain for 30 minutes. If bands are not visible, stain longer. Often several hours of staining produce the best results (overnight staining produces a dark background, but bands will still be visible). Pour the staining solution back into the bottle, (put a check mark on the bottle, for stain is reusable a limited number of times) and add ~300 ml of distilled water to the gel in the tray. The DNA bands should be visible as the background clears. Change the water two or three times. Scientific results need to be documented in permanent form. A photograph of the gel could be used as evidence in the courtroom (the gel itself would break down with age). Set the camera for f22, 1/125 second. Photograph your gel using Polaroid type 667 film. If the photograph is dark change the f stop to f16. If the photograph is light, change the f stop to f32. Results To interpret the size of each of your HindIII bands compare the spacing between the stained bands visible on your gel to the figure given below. The figure gives the expected banding pattern for the size standard and the size of each fragment (the smallest band may not be visible on your gel). a. From your gel photo (or from the gel itself) measure the distance from the well to each of the bands. b. For each of the 4 bands, plot the distance migrated from the well on the X-axis and the size of the DNA fragment on the log scale (Y-axis). Connect the points with a smooth curve. This is a standard curve and can be used to determine the size of the linear fragments in samples treated with restriction enzymes. c. Compare the distance the DNA bands from your PCR reactions migrated to the standard curve to determine their lengths. Analysis 1. Did all of your group's PCRs produce bands? If not, which ones did not? 2. What does this tell you about the identity of your bacterial culture? 3. There are three temperatures in a PCR cycle. In the space below, describe the purpose of each of the steps. 95 degrees Celsius 50 degrees Celsius 72 degrees Celsius There are several components in a PCR reaction. In the space below, describe the purpose of each component in the reaction. 4. DNA polymerase 5. Deoxynucleotide Triphophates (dNTP's) 6. Oligonucleotide primers 7. DNA template 8. Buffer