Lab Procedure 1. 10. ll. 12. 13. First, you will prepare a sample of known tiger DNA that you will compare your unknown samples to. (Notice that your unknown samples are contained in four microcentrifuge tubes in the tube rack. Roll over the tubes to see which DNA samples they contain.) Drag an empty microcentrifuge tube from the Empty Tubes jar to the tube rack. Hover over the tube, and then click in the blank gray box on the face of the tube rack so you can label the tube. Use ?Tiger DNA" or something similar as your label. In the Species Selector, the tiger will already be selected for you. Click on the pipette icon below the species list. You now have a pipette loaded with tiger DNA. Hover over your newly labeled Tiger DNA tube until the pipette straightens up. Click to put the DNA into the tube. Once the pipette is leaning to the right, you?ll know that the DNA is in the tube. (For help using the pipette, watch this video.) Now you will add PCR reagents to your Tiger DNA tube. Do this by clicking on the Taq jar, which will give you a loaded pipette. Then hover over the tube and click (as you did with the DNA pipette) to insert the reagent into the tube. Do the same with dNTPs tag. (Make sure you do not add ?dNTPs.") Note: These PCR reagents have already been added to the other four tubes containing your unknowns. You will now add primers to each of your samples that are specific to the ATP6 gene, which will serve as the point of comparison between your tiger DNA sample and the DNA extracted from the unidentified samples. Click on the small projector behind the tube rack to open the Primer Controls. For each of the primers shown below, copy and paste them one at a time into the blank field at the bottom of the primer selector. Click Add for each one. Now they will appear at the top of the primer menu with asterisks (*). Left primer atgaacgaaaatctattcacc Right primer ttaagtattatcatgtaa Select one of the primers you just added to the list, click the pipette icon beneath the list, and use the loaded pipette to add that primer to the first tube in the rack. Then reload the primer pipette and add it to the next tube. (To reload, simply click the pipette?s tip on the pipette icon below the list of primers.) Repeat until you have added the first primer to all of the tubes. Repeat with the second primer. Be sure to add both primers to all of the tubes. Finally, use PCR to amplify the ATP6 gene from each DNA sample. Drag each tube into the PCR machine. Close the lid and click the green arrow to start the machine. This process, called polymerase chain reaction (PCR), makes multiple copies of the particular gene you are targeting ?in this case, ATP6. (For help running the PCR, watch this video.) PCR takes a while, so click on the arrows on the main lab clock to advance time by a little more than 3 hours. You will now see that the PCR reads ?complete," and that 35 cycles have been performed. Drag the tubes back to the tube rack. Describe what is contained in each of your sample tubes at this point. ANSWER:
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