MD105 Forensic DNA Fingerprinting Lab Exercise PDF

Kyriakou TC. /Markantoni D./Andreou M. F2024 MD105 Cellular and Molecular Biology Lab exercise #3: Forensic DNA Fingerprinting In this lab activity, students will compare band patterns produced by restriction enzyme cleavage of DNA samples when separated on an agarose gel (RFLP). The patterns in th...

Kyriakou TC. /Markantoni D./Andreou M. F2024 MD105 Cellular and Molecular Biology Lab exercise #3: Forensic DNA Fingerprinting In this lab activity, students will compare band patterns produced by restriction enzyme cleavage of DNA samples when separated on an agarose gel (RFLP). The patterns in this exercise are produced from one sample that represents DNA taken at the crime scene and five samples obtained from suspects in the case. Materials/Equipment: 1. Use of the Forensic DNA Fingerprinting Kit #1660007EDU for enzymatic cleavage of DNA 2. DNA Samples 3. EcoRI/PstI restriction enzyme mix 4. HindIII lambda digest (DNA standard) 5. Agarose powder 6. Balance 7. TAE Buffer 8. Agarose Gel Electrophoresis System 9. Sterile Water 10. Waterbath 37C 11. Microwave oven 12. UV Exposure Device 13. DNA Sample Loading Dye 14. Gel Staining Tray 15. Fast Blast DNA Stain 16. Colored microcentrifuge tubes: green, blue, orange, violet, pink, yellow 17. Power Supply 18. Micropipettes and tips 1  Kyriakou TC. /Markantoni D./Andreou M. F2024 EXPERIMENTAL PROCEDURE PART A: Restriction Digestion 1. Place the tube containing the restriction enzyme mix, labeled ENZ, on ice. 2. Label one of each colored microcentrifuge tubes as follows: green tube CS (crime scene), blue tube S1 (suspect 1), orange tube S2 (suspect 2), violet tube S3 (suspect 3), pink tube S4 (suspect 4), yellow tube S5 (suspect 5). 3. Using a fresh tip for each sample, pipet 10 µl of each DNA sample from the stock tubes and transfer to the corresponding-colored microcentrifuge tubes. Make sure the sample is transferred to the bottom of the tubes. 4. Pipet 10 µl of enzyme mix (ENZ) into the very bottom of each tube. Use a fresh tip to transfer the ENZ sample to each tube. Pipet up and down carefully to mix well. 5. Tightly cap the tubes and mix the components by gently flicking the tubes with your finger. If a microcentrifuge is available, pulse spin in the centrifuge to collect all the liquid in the bottom of the tube. Otherwise, gently tap the tube on the tabletop. 6. Incubate the tubes for 45 min at 37°C waterbath. After incubation, proceed to step 7. 7. Using a separate tip for each sample, add 5 µl of loading dye "LD" into each tube. Cap the tubes and mix by gently flicking the tube with your finger. Collect the sample at the bottom of the tube by tapping it gently on the table or by pulse-spinning in a centrifuge. Now your DNA samples should contain: PART B: Agarose Gel Electrophoresis and Visualization of DNA Fragments 1. Weigh out the appropriate amount of agarose(1%) in a flask (1gr). 2. Sprinkle the agarose onto the surface of the TAE in the flask (Add 100mL 1 x TAE Buffer). Note: the agarose will not dissolve until it is heated. 2  Kyriakou TC. /Markantoni D./Andreou M. F2024 3. Dissolve the agarose by heating the solution for intervals of 15-20 seconds in a microwave oven. Remove the flask and gently swirl it around a bit to disperse the contents. 4. Allow the agarose solution to cool until you can comfortably touch the flask with your hands. Add Gel Red staining solution at the flask and mix it. 5. Pour the gel. Place the sample comb in place. Do not move the casting platform until the gel sets. Allow the gel to cure for about 1 hour after it sets. 6. When the agarose gel has solidified, sample loading and electrophoresis can begin. 7. Check that the wells of the agarose gels are near the black (–) electrode and the bottom edge of the gel is near the red (+) electrode. 8. Using a separate tip for each sample, load the indicated volume of each sample into 7 wells of the gel in the following order: Lane 1: S, DNA size standard, 10 µl Lane 2: CS, green tube, 20 µl Lane 3: S1, blue tube, 20 µl Lane 4: S2, orange tube, 20 µl Lane 5: S3, violet tube, 20 µl Lane 6: S4, red tube, 20 µl Lane 7: S5, yellow tube, 20 µl 9. Turn on the power and electrophorese your samples at 120 V for 30 minutes. 10. When the electrophoresis run is complete, turn off the power and remove the top of the chamber. Carefully remove the gel and tray from the gel box. Be careful — the gel is very slippery. Slide the gel into the staining tray. 11. Transfer the gel to the UV Chamber. Observe the banding pattern. 12. Take a picture of the gel and compare the pattern between the samples to reveal the murderer. 3