Lab 6: Principles of PCR Crime Scene Kit PDF

Lab 6: Principles of PCR Crime Scene Kit CHEM 470 Instructor: Randy Wadkins Background The polymerase chain reaction (PCR) is a widely used technique to amplify DNA. While it is used across many scientific disciplines...

Lab 6: Principles of PCR Crime Scene Kit CHEM 470 Instructor: Randy Wadkins Background The polymerase chain reaction (PCR) is a widely used technique to amplify DNA. While it is used across many scientific disciplines, PCR is particularly relevant for forensic DNA analysis as many forensic evidentiary samples contain only trace amounts of DNA. Using PCR, forensic analysts can amplify the small amounts of DNA present in these samples. The resulting amplified DNA can then be used to generate a full DNA profile. At its heart, PCR uses repeated cycles of temperature fluctuations to drive the biochemical reactions that copy DNA.1 In this lab, students will use PCR to prepare mock crime scene samples for real time PCR. After amplifying their samples, students will analyze their PCR products using gel electrophoresis. Students will report the results of this analysis and discuss what those results mean for each suspect in their lab report. Instrumentation and Materials iQ SYBR Green Supermix (MMP) Capless PCR tube adaptors Crime Scene DNA Variable volume micropipette Suspect A DNA Pipette tips Suspect B DNA Thermocycler Suspect C DNA E-gel Power Snap Electrophoresis Suspect D DNA System DNA free water E-gel EX 1% Agarose Gel 5x Loading Dye PCR tubes (strip of six per group) Procedure Day 1 1. From left to right, label your PCR tubes on the sides as NC (Negative Control), CS (Crime Scene), A (Suspect A), B (Suspect B), C (Suspect C), D (Suspect D). Add your group number to the last tube. It is crucial that you label and prepare your samples in this exact orientation. If you label the tube caps, the instrument will not be able to measure the fluorescence of your sample. Make sure that you are wearing gloves when you label your tubes. Try not to lean over or breath into them. 2. Prepare your negative control by added 12.5 μL of DNA free water to the NC PCR tube. 3. Add 12.5 μL of Crime Scene DNA to the labeled PCR tube. Cap the microcentrifuge tube.  4. Repeat step three for the remaining suspect samples. Be sure to only work with one suspect sample at a time and change pipet tips after each use. Each lab member should prepare at least one suspect sample. 5. Using a fresh pipet tip, add 12.5 μL of iQ SYBR Green Supermix (MMP) to the NC labeled PCR tube. Pipette up and down to mix. Note: Be sure not to press the plunger down past the first stop when mixing your samples. Doing so will cause the excess sample to remain in the pipet tip and contaminate other samples. 6. Repeat step 5 with the remaining PCR tubes. Make sure that you are using a fresh pipet tip for each sample. 7. Cap your PCR tubes and place on ice. 8. Once all groups are ready, place your samples in the quantitative PCR thermocycler. Be sure to write down where you placed your samples, as sometimes the samples’ labels can come off during PCR. 9. The TA will start the PCR reaction. Be sure to record the parameters used: Step Function Temperature Time Number of Cycles Initial Denaturing Denature 94°C 2 min 1 Thermal Cycling Denature 94°C 30 sec 35 Anneal 52°C 30 sec Extend 72°C 1 min Melt Curve 55°C-95°C heat at 0.5°C, 1 Analysis measure every 10 sec 10. Based on the remaining time, Dr. Wadkins will determine whether to continue the lab or allow the PCR to run overnight. If time permits, proceed directly to the day 2 procedure. Day 2 1. Set up your electrophoresis units. If running the entire experiment on the same day, do not open your gels until you have your samples ready to load. 2. Collect your group’s PCR tubes from the thermocycler. Place each sample in a capless microcentrifuge tube adaptor and pulse spin to collect all liquid to the bottom of the tube. 3. Dilute each sample according to the TA’s instructions on the board. 4. Add 1 μL of loading dye to each PCR tube. Remember to use a fresh pipet tip for each sample. 5. Open your gel and place it into the electrophoresis unit. 6. Load 20 μL of each sample onto the gel. By convention, your allelic ladder should be loaded in the first well. Load the rest of your samples in the following order: NC, CS, A, B, C, and D. 7. Select the appropriate method to run your gels. Be sure to write this down as you will need to include it in your lab report. Keep an eye on your gel as it is running to prevent samples from running off the edge of the gel. 8. Use the imaging unit to take and save a picture of your gel. References (1) Li, R. Forensic Biology, 2nd ed.; CRC Press: Boca Raton, FL, 2015.