PCR-Based Crime Scene Analysis Lab

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Questions and Answers

What is the purpose of using a fresh pipet tip for each suspect sample?

To speed up the mixing process

To prevent cross-contamination between samples (correct)

To ensure consistency in sample volume

To increase the temperature of the samples

What is the first step in the PCR reaction setup as described?

Place the samples in the thermocycler

Cap the PCR tubes and place them on ice

Add iQ SYBR Green Supermix to the PCR tubes (correct)

Add loading dye to the PCR tubes

What should not be done when mixing samples with the pipet?

Use the same pipet tip for multiple samples

Cap the tube before mixing

Press the plunger down past the first stop (correct)

Mix the sample for too long

How long should the initial denaturing step last during the PCR process?

2 minutes Signup and view all the answers

What is the temperature used for the annealing step during thermal cycling?

52°C Signup and view all the answers

What should be recorded before placing the samples in the quantitative PCR thermocycler?

Where the samples are placed in the thermocycler Signup and view all the answers

What is the purpose of pulse spinning the microcentrifuge tube after collecting PCR tubes from the thermocycler?

To collect all liquid to the bottom of the tube Signup and view all the answers

What should be done with the gel before loading samples in electrophoresis?

Open the gel after preparing the samples Signup and view all the answers

What is the primary purpose of PCR in forensic analysis?

To amplify trace amounts of DNA for profiling Signup and view all the answers

Why is it important to label the sides of the PCR tubes rather than the caps?

To ensure the instrument can measure fluorescence accurately Signup and view all the answers

What role does DNA free water play in the PCR process?

It serves as a negative control sample Signup and view all the answers

Which of the following materials is used for analyzing PCR products?

E-gel Power Snap Electrophoresis System Signup and view all the answers

What steps should be taken to prevent contamination when preparing PCR samples?

Wearing gloves and avoiding breathing into the samples Signup and view all the answers

In the PCR process, what is the significance of using repeated cycles of temperature fluctuations?

To drive the biochemical reactions that copy DNA Signup and view all the answers

How many μL of Crime Scene DNA should be added to its respective PCR tube?

12.5 μL Signup and view all the answers

What is the primary function of the iQ SYBR Green Supermix in the PCR process?

To contain the necessary reagents for DNA amplification Signup and view all the answers

Study Notes

PCR-Based Crime Scene Analysis

This lab simulates crime scene analysis using PCR, a technique amplifying trace DNA amounts.
Students will use the technique to generate and analyze mock crime scene samples, mimicking real-world forensic investigation.
Materials:
- DNA Samples: Crime scene, four suspects (A-D), and DNA-free water for negative control
- PCR Reagents: iQ SYBR Green Supermix (MMP) - a reagent that fluoresces when bound to DNA, enabling real-time PCR analysis.
- Equipment: Micropipettes, pipette tips, PCR tubes, thermocycler, E-gel electrophoresis system with 1% Agarose Gel, and loading dye.

PCR Procedure

Carefully label PCR tubes to avoid sample mix-up: NC (Negative Control), CS (Crime Scene), A (Suspect A), B,C, D, and a tube labeled with the group number. Ensure no labels are on the tube caps, which can interfere with fluorescence measurement.
Sample Preparation: Add 12.5 μL of each DNA sample (Crime Scene and Suspects) to their respective labelled PCR tubes.
Negative Control: Add 12.5 μL of DNA-free water to the NC tube.
Master Mix Addition: Add 12.5 μL of iQ SYBR Green Supermix (MMP) to each tube, ensuring separate tips for each sample to prevent contamination.
Thermocycling:
- Initial Denaturation: 94°C for 2 minutes, one cycle.
- Thermal Cycling: 35 cycles of:
  - Denaturation: 94°C for 30 seconds.
  - Annealing: 52°C for 30 seconds.
  - Extension: 72°C for 1 minute.
- Melt Curve Analysis: 55°C to 95°C, heating at 0.5°C every 10 seconds for data measurement.
Day 2 - Electrophoresis:
- Set up the electrophoresis unit and gels.
- Sample Preparation: Collect PCR tubes, pulse spin to collect liquid, and dilute each sample as instructed.
- Loading: Add 1 μL of loading dye to each sample.
- Gel Loading: Carefully load 20 μL of each sample onto the prepared gel.

Understanding Lab Results

Students analyze their PCR results by comparing DNA profiles generated from the crime scene sample to the suspects.
This might involve matching specific DNA bands or patterns to determine which suspect's DNA matches the crime scene sample.
Results and their interpretation are crucial components of the lab report.

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Description

This lab quiz simulates real-world forensic DNA analysis using PCR techniques. Students will engage in a mock crime scene investigation by analyzing DNA samples from suspects and a crime scene. The activity involves using various PCR reagents and equipment to generate accurate results.