ࡱ> TVS 0bjbj ;R՟՟(BBBBBVVVVrlVRTTTTTT$KzxBxiBBiiiBBRiRiiVv| Q*䒕R >0 p iB 4xxi* :   DNA Fingerprinting: A Simulation Name_______________________________________Period____Tray #____Date______________ Team Members: Dumas/05 The Scenario: You are a lab team at a forensics lab. Detectives have brought you a blood sample from a crime scene to analyze the DNA. They have also brought you blood samples from 3 suspects, each of whom had probable cause and opportunity to commit the burglary. It is up to your crack team to solve the mystery! In this lab we will be simulating the DNA techniques using bacterial DNA rather than human DNA because bacterial DNA is simpler. Obviously the three suspects have different DNA (coming from different bacteria). Only one of them matches the crime scene DNA. Procedures for DNA Fingerprinting using Bacterial DNA Rational: In this exercise you will receive DNA from three different bacteria. They will be labeled as Suspect 1, Suspect 2, and Suspect 3. You will also receive Crime Scene DNA that will be of the same type as one of your 3 Suspects (maybe). You will also receive the Restriction Enzyme named EcoRI. When this enzyme cuts up the bacterial DNA, the circular DNA will be cut open into a straight piece. Since not all bacterial DNA are the same length, electrophoresis could cause them to travel different distances through a gel. Furthermore, the EcoRI could cut the bacteria in more than one place causing several pieces to result. In this case each piece of a different length would travel different distances. The pattern in the gel will allow you to determine which suspect has the same DNA as the DNA found at the crime scene. Does matching DNA prove guilt . . .that decision belongs to the jury. You can only report your findings and let the jury decide. In the description above: What is the experimental question? What is the manipulated variable? What is the responding variable? What other variables must be controlled? To improve the quality of your data, you will analyze all of the data from the lab teams. Your data presentation, analysis and conclusions along with evaluation will be written as though you had been called to court as an expert witness to respond to both the prosecutor and the defense counsel during a trial. You will report on behalf of your entire lab staff. (Be sure to use your IB Criteria when preparing for courtthe same standards apply.) Prosecutor: "How does DNA fingerprinting work?" (5) "Who did it?" Be very careful on this question! (2) "How do you know?" (4) Defense Counsel: "What kinds of errors could have occurred?" (4) "With these problems, how can you be sure that you have named the correct person?" (2) Materials & Equipment: Shared by the whole class 37oC water bath microcentrifuge 1X TAE running buffer (1000 mL) gel electrophoresis box power supply ethidium bromide staining bath UV transilluminator No. 667 polaroid film DNA stain on ice 1 kb DNA ladder EcoRI (2 L per reaction) 2.0 L of Crime Scene DNA 2.0 L of DNA from Suspect 1 2.0 L of DNA from Suspect 2 2.0 L of DNA from Suspect 3 per group micropipettors (p-20) racks of tips for pipettors six empty microfuge tubes one microfuge tube rack one marking pen microfuge tube with distilled H2O ruler calibrated in mm (1 per student) half of an 8% agarose gel (12 wells) 4.0 L of gel loading dye crushed ice in insulated cup 35.0 L distilled water microfuge tube float jar for tip disposal  ***The quality of experimental results depends upon the quality of your reagents. Restriction enzymes must be treated with special care! Since they are unstable at higher temperatures, all enzymes are stored at -20oC and kept on ice during use. Procedure: I. Prepare Restriction Digest (DNA Cutting) 1. Label four 1.5 microfuge tubes with symbol &: Crime Scene, Suspect 1, Suspect 2, Suspect 3, etc 2. All reagents must be stored on ice, but the reaction tubes may be kept at room temperature in the tube rack at the lab station. 3. Use the matrix below as a guide when you add the reagents to each tube. Reagents must be added in order or results will be non-existent. 4. Go to the DNA Station. Using a micropipettor, add 5.0 L of DNA to appropriately labeled reaction tube. Touch the pipette tip to the side of the tube, as near the bottom as possible to create capillary action to pull the solution out of the tip. 5. Go to the Enzyme Station. Use fresh tips to add 5.0 L of EcoRI to the tubes as shown in the table. Put samples into the ice cup as soon as you have added the enzyme. MATRIX TubeDNAEcoRICrime Scene5.0 L 5.0 LSuspect 15.0 L 5.0 LSuspect 25.0 L 5.0 L S Suspect 35.0 L 5.0 L  6. Close the caps and spin the tubes for a 1-2 second pulse in the microcentrifuge. Make sure the tubes are placed in a balanced configuration in the rotor. This will pool and mix the reactants in the bottom of the tube. Place the tubes in the 37oC waterbath for 45 minutes of incubation time. Rinse the tips in the disposal jar. Blow them dry ***Stopping point***The reaction tubes may now be placed in a refrigerator until you are ready to continue, or you may proceed immediately with the electrophoresis and staining procedures. II Electrophoresis to separate pieces of cut DNA 1. Set an empty gel bed so that one end of the bed has the end up and tightly screwed while the other end is down. 2. Take the agarose gel previously prepared and stored in a baggy in the refrigerator. Add more 1XTAE Buffer to the baggy so that the gel will slip out of the baggy onto the gel bed. Slide it onto the gel bed through the down end of the bed. Make sure that the gel has its upper side up so that the holes are on top. Be careful not to break or damage the gel or two teams will not be able to proceed. Put the 1XTAE buffer that is still in the baggy into the electrophoresis chamber. 3. Carry the gel bed to the electrophoresis chamber. Carefully loosen the screws at the up end of the gel bed and lower the end. Retighten. 4. Place the tray on the platform of the gel box, so that the comb is at the negative (black) end. 5. Fill the electrophoresis chamber with 1XTAE buffer to a level that just covers the entire surface of the gel. (TAE buffer can be used several times, so do not discard it.) 6. Double check to make certain that the well holes are completely submerged. If you notice "dimples" around the well holes, slowly add buffer until they disappear. 7. Remove the reaction tubes from the refrigerator and let reaction tubes warm to room temperature. Holding them in warm hands is an acceptable procedure as 37oC is an acceptable temperature. 8. Add 2 L of loading dye to each reaction tube. This dye contains a DNA stain. 9. Close caps and spin tubes for a 1-2 second pulse in the microcentrifuge making sure that the tubes are placed in a balanced configuration in the rotor. This mixes the loading dye and DNA stain with the reactants. 10. Use a micropipettor to load the contents reaction tube A into a well on the gel. --Set the micropipet and extract all 10.0 L from Reaction Tube labeled Crime Scene. --Using 2 hands, steady the pipet over the well. --Be careful not to punch the pipet through the bottom of the gel. --Gently depress the pipet plunger to slowly expel the sample into the appropriate well. If the tip is centered over the well the reaction solution will sink to the bottom of the well. Keep the plunger depressed to the second stop until you have removed it from the fluids. This will keep you from removing the well contents. 11. Load a ladder with 2 L of loading dye into the well between the two teams. This will later be used for measuring the lengths of the DNA pieces in the fingerprint. 12. Repeat step 10 with each of the tubes of DNA from the suspects 13. On the diagram below, indicate the wells where each of your reaction tube contents was placed. outside center ladder  14. Close the top of the tank, and connect electrical leads--red to red, and black to black. Make sure both electrodes are connected to one channel of the power supply. 15. Set the power source on 100 volts and turn the unit on. Check the ammeter which should register 50-100 milliamps. This confirms that current is flowing through the gel. If you do not detect a current, check the connections and try again. Caution: Electric shock hazard! Do not put fingers or other objects into the buffer solution while the power supply is on!!! 16. Shortly after the current is applied, you should see the dye moving through the gel toward the positive end of the gel box. It will appear as a blue band. Eventually this band will resolve into two bands--one turquoise and one purple. 17. Run the electrophoresis for 45 minutes. Good separation of the fragments will have occurred when the first dye front has moved 4-8 cm from the well. Rinse & blow dry the used tips. 19. When the dye has moved sufficiently, turn off the power supply. Then disconnect the leads. 20. Carefully remove the gel from the electrophoresis chamber. ***Stopping point***Once the loading dyes have separated out of the well about 1 cm, the electrophoresis may be stopped and the gel stored in a baggy in the refrigerator for 24 hours. Then electrophoresis may be resumed until the dyes have moved sufficiently to get a good spread of DNA. III Viewing: 1. Gently move the gel to a UV transilluminator. Turn on the illuminator and look for bands of DNA. If there are bands on the gel you may now take a picture with a polaroid camera. The picture can be xeroxed to provide multiple copies for others on your team. IV. Gel Interpretation: 1. What do your results show? 2. Did you get any unexpected results? Describe them. 3. What is a 1 Kb DNA ladder? How was it used in these procedures? 4. Make a Data Transformation Table on a separate piece of paper or on a computer that shows the DNA label, the number of pieces of DNA that resulted from the digest reaction, the distance traveled by each piece and the length of each piece in base pairs. 5. Make a graph of the known lengths of DNA in the ladder against the distances traveled by these pieces. Use the line of best fit of the ladder to refine the rough estimates of your suspect's DNA pieces. 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