Frensic Science Grup: Backgrund infrmatin One f the graduate students in the Department f Bilgy n the campus f the University f Flrida went missing. A week later, a crime scene was discvered n a remte part f campus. The bdy was fund wrapped in a blanket and tied up with a vine later identified as a species f Vitis. Fiber samples and plant material was cllected frm the crime scene. Based n the investigatin, fur suspects were identified as pssibly being invlved. Each suspect had his/her persnal effects searched and cllected. Each grup is supplied with cllected evidence and charged with trying t find ut which suspect(s) is respnsible fr the murder. T d this, three pieces f evidence will be used: leaf mrphlgy f plants cllected, DNA evidence f plant material, and fiber analysis. Leaf Mrphlgy In the space prvided please describe aspects f the leaf material recvered frm the crime scene and the suspect. Sme examples f things t fcus n: Cmparative leaf size 1
Overall shape Edge f leaf (smth versus teeth) Single leaf r cnnected Directin f leaf veins Anything else that stands ut 2
Describe the leaf material frm the Crime Scene Describe the leaf material frm 1 Describe the leaf material frm 2 Describe the leaf material frm 3 Describe the leaf material frm 4 D any f the leaves frm suspects lk like the Crime Scene sample? Based just n leaf mrphlgy, wh d yu think cmmitted the crime? 3
DNA Extractin 1. Rinse frceps and scissrs with 70% ethanl prir t use and between handling different samples 2. Cut a rughly 0.5 cm wide piece f leaf material frm a leaf, r use a hle punch t btain a leaf disc. Place int a 2 ml cllectin tube. Repeat t have a ttal f 5 extractins frm the crime scene and all f the suspects. 3. Add 100 µl f Extractin Slutin t the cllect tube. Clse tube and vrtex briefly t mix. Make sure leaf material is cvered by slutin. 4. Incubate at 95 C fr 10 minutes. Leaf tissue will nt appear t be degraded. 5. Add 100 µl f Dilutin Slutin and vrtex briefly t mix. Remval f leaf material is nt necessary. 1. Find REDExtract-N-Amp Ready Mix, water, frward and reverse at yur wrk statin. Which pairs des yur grup have? 2. Each reactin will cntain the fllwing quantities: 10 µl REDExtract-N-Amp Ready Mix 2 µl f Frward Primer 2 µl f Reverse Primer 2 µl f water 4 µl f leaf extract 20 µl ttal reactin 3. Calculate the amunt f reagents needed t cnduct fr the 5 extractins + a negative cntrl (ttal f 6 reactins). Reagent Ready Mix Ttal amunt needed fr Master Mix Frward Primer Reverse Primer Water 4
4. Make a master mix fr each pair that yu received by adding the apprpriate reagents int a 1.5 eppendrf tube. After each reagent is added, check it ff n the list belw t ensure each has the apprpriate material Primer Pair Ready Mix Frward Primer Reverse Primer Water Primer Pair Ready Mix Frward Primer Reverse Primer Water 5. Befre adding yur master mix t the tubes, add in 4 µl f DNA extract int a labeled tube. Yu will need 2 tubes fr each DNA sample, since we are running tw different markers fr each sample. After all f the DNAs are added, and water fr the negative cntrls, add in yur made up master mix. Yu will need t add 16 µl f this mix t each tube. 6. Mix briefly and then centrifuge t cllect all cmpnents at the bttm f the tube. 7. Place in thermcycler and run under the fllwing cnditins: Initial denaturatin at 95 C fr 3 minutes 35 cycles f Denaturatin at 95 C fr 30 secnds Annealing at 50 C fr 30 secnds Extensin at 72 C fr 45 secnds Final extensin at 72 C fr 10 minutes Hld at 4 C indefinitely 5
Prepare gel 1. Plug PwerBase int an electrical utlet. 2. Remve gel cassette frm package 3. Insert the gel (with cmb in place) int the base right edge first. The Invitrgen lg shuld be lcated at the bttm f the base. Press firmly at the tp and bttm t seat the gel cassette in the PwerBase. A steady, red light will illuminate if the gel cassette is crrectly inserted. Lad prepared samples Well # 1 2 3 4 5 6 7 8 9 10 11 12 What t add t the well Crime Scene first 1 first 2 first 3 first 4 first Negative cntrl first Crime Scene secnd 1 secnd 2 secnd 3 secnd 4 secnd Negative cntrl secnd 1. Remve and discard cmb frm the E-Gel cassette. 2. In the first 6 wells, lad gel by adding 20 µl f prduct f their first t each well. Prduct f the secnd will be added in wells 7-12. Run gel 1. Press and release the 30 minute buttn n the E-Gel PwerBase t begin electrphresis. 2. At the end f the run, the current will autmatically shut ff and the pwer base will display a flashing red light and beep rapidly. Press either buttn t stp the beeping, and unplug the E- Gel PwerBase. 3. Remve the gel cassette and analyze yur results by viewing n ne f the transilluminatrs. 6
Gel Results What d yu see when yu visualize the gel? What des the Crime Scene sample lk like? D any f the suspect samples lk similar t the Crime Scene sample? If s, which ne(s)? Based nly n the DNA results, wh d yu think cmmitted the crime? 7
Hair and Fiber samples Remving Fibers frm Cllect Tape 1. Place the cllectin tape int a beaker with arund 400 ml f water. The evidence frm this tape was cllected frm crime scene n the UF campus. 2. Gently agitate the tape t disslve the adhesive. 3. When the fibers have detached, remve them frm the beaker and place n paper twel/kimwipe t dry. Hw many samples d yu have frm the Crime Scene? Hw many samples d yu have frm 1? Hw many samples d yu have frm 2? Hw many samples d yu have frm 3? Hw many samples d yu have frm 4? Preparing slides 1. Paint a small amunt f casting medium n t the center f the slide. 2. Place a hair r fiber sample int this drp f casting medium. The casting medium will act as a glue t permanently attach the strand t the slide. 3. Carefully place a cver slip ver the strand and medium. Press gently n the slip t ensure the cverslip seals cmpletely. 4. Label the slide with the apprpriate persn it came frm (crime scene r which suspect). 5. Repeat until yu have all hair and fiber samples prepared. 6. T visualize the slide, place nt micrscpe stage. Adjust and fcus the scpe t examine the munted stranded. MAKE SURE TO START OUT AT 4X, GET THE MATERIAL INTO FOCUS, THEN MOVE UP TO A HIGHER MAGNFICATION IF NECESSARY. 8
7. Recrd ntable characters f each sample such as: Is the sample hair r fiber? Clr? Shape? Describe samples frm crime scene. Describe samples frm 1. Describe samples frm 2. Describe samples frm 3. Describe samples frm 4. D any f the samples frm the suspect match thse fund at the crime scene? Based just n the fibers, wh d yu think cmmitted the crime? 9
Overall results Based slely n leaf mrphlgy, wh is the likely criminal? Based slely n DNA, wh is the likely criminal? Based slely n the fiber analysis, wh is the likely criminal? D all three pieces f evidence tell the same stry? If nt, hw d they differ? Based n all f the data cmbined, which suspect (if any) wuld yur grup charge with the crime? 10